M.V. Hartog

The Arabidopsis somatic embryogenesis receptor kinase 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Somatic embryogenesis from Arabidopsis shoot apical meristem mutants.

Abstract. Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.

Purification, immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells.

A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.

CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes

Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.

Facilitated Initiation of Somatic Embryogenesis in Arabidopsis Thaliana by Mutations in Genes Repressing Meristematic Cell Divisions

A very efficient and reproducible system for somatic embryogenesis in Arabidopsis was established by using intact seedlings of the primordia timing mutant (pt). Embryogenie clusters originated from the enlarged shoot apical meristem (SAM) of the mutant seedlings when germinated in 2,4-D containing liquid media, pt somatic embryos had all characteristic embryo pattern elements, but with higher and more variable numbers of cell layers and cells per cell layer. This finding shows that pattern formation can be completed in somatic embryos without the regular cell division pattern seen in zygotic embryos. Embryogenie cell lines were also established from seedlings of other mutants with enlarged SAMs, such as clavatal and clavata3 (civ), pt civ 1-4 and pt clv3-2 double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenie cell lines. This data suggest that an increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

Cell Wall Glycoprotein Encoding Genes in Somatic and Zygotic Embryogenesis

In carrot, somatic embryos can develop after simple culture manipulations from single embryogenic cells (Komamine et al. 1990) or from clusters of embryogenic cells designated proembryogenic masses (Halperin 1966). Most embryogenic carrot cultures are maintained over many subcultures with 2,4-D either as the sole growth regulator, or in the presence of both 2,4-D and cytokinin. Therefore, cells that have embryogenic potential may either be continuously formed from non-embryogenic cells, or constitute an independent self-propagating subpopulation. In carrot cultures, depletion of the population of embryogenic cells eventually leads to loss of the embryogenic potential. In Figure 1 a schematic representation of the acquisition and expression of embryogenic potential in carrot cultures is presented. In other culture systems such as alfalfa, non-embryogenic cells can be maintained in media containing NAA, while a short exposure to a high concentration of 2,4-D is sufficient for these cells to acquire embryogenic potential (Bogre et al. 1990). It is unlikely that 2,4-D is unique in its role in the establishment of embryogenic potential. For instance, Smith and Krikorian (1990) have described a culture system in which cells derived from carrot zygotic embryos maintain their embryogenic potential indefinitely in the absence of any growth regulator, solely by subtle adjustments of the medium pH. In carrot, it is not simply the removal of 2,4-D that triggers embryo development, but rather the change in cell density that appears to allow the formation of globular embryos, even in the presence of 2,4-D.