Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

The petunia MADS box gene FBP11 determines ovule identity.

In contrast to the wealth of information relating to genes regulating floral meristem and floral organ identity, only limited data are available concerning genes that are involved in determining and regulating the identity and development of an ovule. We have recently isolated the floral binding protein 11 (FBP11) MADS box gene from petunia and found that it is expressed exclusively in ovule primordia and subsequently in the ovules, suggesting a role for this gene in ovule formation. To test this hypothesis, we constructed a recombinant gene in which the full-size FBP11 cDNA was placed under the control of a strong cauliflower mosaic virus 35S promoter. Transgenic petunia plants expressing this chimeric gene have ovulelike structures on the adaxial side of the sepals and the abaxial side of the petals. Detailed morphological studies showed that these ovulelike structures are true ovules. RNA gel blot analysis was performed to investigate ectopic FBP11 expression in relation to the expression of the closely related FBP7 gene and the putative petunia class C-type homeotic genes FBP6 and pMADS3. Our results indicate that FBP11 represents an ovule identity gene. A new model describing the mode of action of FBP11 as an additional class D MADS box gene is presented.

A novel class of MADS box genes is involved in ovule development in petunia.

We isolated and characterized two ovule-specific MADS box cDNAs from petunia, designated floral binding protein (fbp) genes 7 and 11. The putative protein products of these genes have approximately 90% of their overall amino acid sequence in common. In situ RNA hybridization experiments revealed that both genes are expressed in the center of the developing gynoecium before ovule primordia are visible. At later developmental stages, hybridization signals were observed only in the ovules, suggesting that these genes are involved in ovule formation. To test this hypothesis, we raised transgenic petunia plants in which both fbp7 and fbp11 expression was inhibited by cosuppression. In the ovary of these transformants, spaghetti-shaped structures developed in positions normally occupied by ovules. These abnormal structures morphologically and functionally resemble style and stigma tissues. Our results show that these MADS box genes belong to a new class of MADS box genes involved in proper ovule development in petunia.

Petunia hybrida homologues of shaggy/zeste-white 3 expressed in female and male reproductive organs.

Petunia hybrida ovule-specific cDNA libraries were screened for genes encoding homologues of protein kinases known to be involved in developmental processes. Two cDNAs, PSK4 and PSK6 (Petunia hybrida shaggy related protein kinase), homologous to GSK-3 from rat, MDS1 from yeast and to the segment polarity gene shaggy/zeste-white 3 (sgg/zw3) from Drosophila were characterized in detail. Southern blot analysis showed that the PSK4 and PSK6 genes belong to a small multigene family in P. hybrida. The PSK4 and PSK6 proteins, which are 70% identical to sgg/zw3 and its functional homologue GSK-3 over the protein kinase catalytic domain and 55% identical to MDS1, represent two different subgroups of protein kinases. RNA gel blot and RT-PCR analyses revealed that the PSK4 gene is expressed during the vegetative phase and the female reproductive phase of development and the PSK6 gene predominantly during the male and female reproductive phases. In situ hybridization on developing flower buds confirmed that PSK4 is expressed throughout the different developmental stages from ovule primordium formation until embryo sac maturation, while PSK6 showed expression during anther cell differentiation and at pollen maturity, and an expression pattern similar to PSK4 in the female reproductive organs. The results also revealed transcripts of a PSK gene in embryos at the globular stage.

A homologue of the MAP/ERK family of protein kinase genes is expressed in vegetative and in female reproductive organs of Petunia hybrida.

The mitogen activated protein (MAP) kinase pathway of eukaryotes is stimulated by many growth factors and is required for the integration of multiple cellular signals. In order to study the function of MAP kinases during plant ovule development we have synthesized a Petunia hybrida ovule-specific cDNA library and screened for MAP protein kinase-related sequences using a DNA probe obtained by PCR. A full-length cDNA clone was identified (PMEK for Petunia hybrida MAP/ERK-related protein kinase) and shown to encode a protein related to the family of MAP/ERK protein kinases. Southern blot analysis showed that PMEK is a member of a small multigene family in P. hybrida. The cDNA codes for a protein (PMEK1) of 44.4 kDa with an overall sequence identity of 44% to the products of the mammalian ERK/MAP kinase gene, and the budding yeast KSS1 and FUS3 genes. PMEK1 displays 96 and 80% identity respectively with the tobacco NTF3 and Arabidopsis ATMPK1 kinases, and only 50% to the more distantly related plant MAP kinase MsERK1 from alfalfa. The two phosphorylation sites found in the loop between subdomain VII and VIII in all the other MAP kinases are also present in PMEK1. RNA gel blot and RT-PCR analyses demonstrated that PMEK1 is expressed in vegetative organs and preferentially accumulated in female reproductive organs of P. hybrida. In situ hybridization experiments showed that in the reproductive organs PMEK1 is expressed only in the ovary and not in the stamen.

Ultrastructural changes of Arabidopsis thaliana pollen during final maturation and rehydration.

Several ultrastructural changes occur during dehydration and subsequent rehydration of Arabidopsis thaliana pollen. The cytoplasmic channels, present in the outer part of the intine of the mature, dehydrating pollen grain, degenerate and develop into electron-dense inclusions. At the same time a large quantity of electron-dense material is deposited in the cavities of the exine. A large number of vesicles is produced in the vegetative cell, and they become predominantly located in the peripheral region near the intine. Starch of amyloplasts is consumed and the lipid bodies which originally surround the sperm cells become randomly distributed. In addition, the individual lipid bodies become enveloped by single rough endoplasmic reticulum cisterns.

Unequal distribution of plastids during generative cell formation in Impatiens.

This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts.

Ultrastructure and histochemistry of Citrus limon (L.) stigma.

Citrus limon has a "wet" stigma which can be divided in two zones: a glandular superficial one formed by papillae, and a non-glandular one formed by parenchymatic cells. The stigmatic exudate is produced by the papillae after the latter have reached their ultimate size. The papillae of the mature pistil are of varying size and composition. Both the unicellular and multicellular ones are present. The cells at the base of the papillae are rich in cytoplasm, whereas the tip cells are vacuolated. Histochemical analysis has shown that the exudate of Citrus is composed of lipids, polysaccharides, and proteins. Our results indicate that the lipidic component is produced and secreted first, followed by production and secretion of the polysaccharidic component. The lipidic component of the exudate is produced in the basal papillae cells and accumulates as droplets in dilated parts of the smooth endoplasmic reticulum (SER). Subsequently the lipid droplets are transported to the plasma membrane, and transferred by the latter into the cell walls. Then the exudate component is accumulated in the intercellular spaces and in the middle lamellar regions of the walls. Subsequently, the polysaccharidic component of the exudate is produced and secreted by the tip cells of the papillae.

Spina bifida and so-called asplenia syndrome occurring separately in sibs.

Attention is drawn to the possibility that neural-tube defects may sometimes be associted with the so-called asplenia syndrome (Ivemark syndrome). This hypothesis is based upon a family in which one child had spina bifida and hydrocephaly and another had cardiovascular and other fissural anomalies, similar to Ivemark syndrome. The family history, moreover, revealed several cases of anencephaly and/or spina bifida, both on the paternal and maternal sides.

Ultrastructure of transmitting tissue of Lycopersicon peruvianum style: Development and histochemistry.

The development of the transmitting tissue of the style of Lycopersicon peruvianum goes, after the completion of cell division and cell wall formation, through two distinct phases. During the first phase, the cells enlarge and the main part of the intercellular substance, consisting of pectins, is formed. During the second phase, the cells form an extensive rough endoplasmic reticulum (RER) and proteins are incorporated in the intercellular substance. A possible role of these proteins in the incompatibility reaction is proposed.

Callose deposition and plug formation in Petunia pollen tubes in situ.

In Petunia pollen tubes growing in the style there appear to be two ways of callose deposition. The first one is callose deposition outside the plasma membrane as a distinct layer closely appressed to the cell wall. The second one is callose deposition within the cytoplasm as distinct callose grains, leading to the formation of callose plugs. This second way is accompanied by a characteristic ultrastructure of the cytoplasm, namely strong electron-density of the plasma matrix, partial absence of the plasma membrane and the absence of plastids and dictyosomes. For both ways of callose deposition a mechanism is proposed and the function of callose plugs is discussed.