William J. Gordon-Kamm

A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD γ-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis.

Cyclin-Dependent Kinase Inhibitors in Maize Endosperm and Their Potential Role in Endoreduplication

Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13Suc1. They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway

The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Using the maize Bbm and Wus2 genes enhances transformation efficiency in maize and other monocots, broadens the genotype range, and permits transformation of mature seed-derived embryos and leaf segments. While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Site-specific recombination for genetic engineering in plants

Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now possible in the course of genetic transformation procedures, thus making the process more predictable and reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying such methods to plants opens new avenues for large-scale chromosome engineering in the future.

Effects of microprojectile bombardment on embryogenic suspension cell cultures of maize (Zea mays L.) used for genetic transformation

We have investigated the interaction between tungsten and gold microprojectiles with suspension-culture cells of maize used for genetic transformation. Particle size measurements were evaluated before and after DNA precipitation to determine mean particle size and the effect of DNA precipitation on particle aggregation. Following particle bombardment, metal foils were examined by scanning electron microscopy to visualize dispersion of individual particles and aggregates. Particle penetration into suspension-culture cell clusters was examined in paraffin-embedded bombarded cells serially sectioned and viewed with light microscopy and by energy dispersive X-ray microanalysis. Acridine-orange-stained bombarded cells were examined to observe cellular response to particle penetration. Transient expression of reporter genes C1 and B and GUS, (β-glucuronidase) were used to assess effects of particle bombardment on embryogenic cell types. Autoradiographic analysis of the transformable suspension cell culture SC82 (see Gordon-Kamm et al. 1990, Plant Cell 2, 603–618) was conducted to evaluate the S-phase and mitotic indices in embryogenic and nonembryogenic cells throughout a subculture passage and in response to DNA/particle delivery. The results of these investigations are discussed relative to cytodifferentiation of suspension cell clusters and recovery of transformed clonal sectors.

Positive regulation of minichromosome maintenance gene expression, DNA replication, and cell transformation by a plant retinoblastoma gene

Retinoblastoma-related (RBR) genes inhibit the cell cycle primarily by repressing adenovirus E2 promoter binding factor (E2F) transcription factors, which drive the expression of numerous genes required for DNA synthesis and cell cycle progression. The RBR-E2F pathway is conserved in plants, but cereals such as maize are characterized by having a complex RBR gene family with at least 2 functionally distinct members, RBR1 and RBR3. Although RBR1 has a clear cell cycle inhibitory function, it is not known whether RBR3 has a positive or negative role. By uncoupling RBR3 from the negative regulation of RBR1 in cultured maize embryos through a combination of approaches, we demonstrate that RBR3 has a positive and critical role in the expression of E2F targets required for the initiation of DNA synthesis, DNA replication, and the efficiency with which transformed plants can be obtained. Titration of endogenous RBR3 activity through expression of a dominant-negative allele with a compromised pocket domain suggests that these RBR3 functions require an activity distinct from its pocket domain. Our results indicate a cell cycle pathway in maize, in which 2 RBR genes have specific and opposing functions. Thus, the paradigm that RBR genes are negative cell cycle regulators cannot be considered universal.

A NOVEL TECHNIQUE FOR THE PARTIAL ISOLATION OF MAIZE EMBRYO SACS AND SUBSEQUENT REGENERATION OF PLANTS

SummaryEmbryo sacs of maize isolated with a few layers of surrounding nucellus or completely isolated with digestive enzymes have resulted in either poorly visible or structurally damaged embryo sacs. We therefore developed a new, more successful method involving mechanical sectioning of maize ovaries using the Vibratome. Sections containing intact embryo sacs are viable and development is normal when sacs are cultured in vitro on semisolid Murashige and Skoog (MS) media. Embryo sacs produce endosperm (90%) and embryos (75%), and mature plants are obtained directly without callus formation or somatic embryogenesis. Immediate applications of this technique may include experimental fertilization and embryogenesis as well as genetic manipulation. Targeting of individual cells was demonstrated with microinjection and confocal microscopy. The methods developed in this study provide a way of studying maize embryo sac development and transformation.

Transgenic Cereals — Zea mays (maize)

Genetic transformation of maize is routine in several genotypes despite the many difficulties encountered in developing reliable transformation techniques in this major cereal species. Aspects of maize tissue culture, including the target expiant, subsequent rapid in vitro proliferation and dependable regeneration from competent cells were prerequisite developments for gene delivery into maize. Recovery of transgenic, fertile maize required high levels of gene expression and identification of new selectable markers, along with DNA delivery into competent maize cells. DNA delivery by particle bombardment, Agrobacterium, electroporation and silica fiber methods have been the most carefully documented, each of which can now be used for gene transfer into maize. Promoters such as those from the CaMV 35S or ubiquitin genes, together with various introns have been widely used to achieve high expression levels, while the herbicide resistance gene, bar, has served as an important selectable marker for numerous studies in maize transformation. Although tissue culture cells were instrumental in the development of maize transformation, the direct use of expiants such as the immature embryo and/or meristems has found favor in more recent applications. Gene delivery in maize has shifted from emphasis on technology development to evaluation of gene expression with various transgenes, some of which are already in large-scale commercial development (e.g. insect and herbicide resistance). Maize transformation is increasingly being used to address more sophisticated aspects of gene regulation, plant development and physiology. The stability of transgene expression in primary transgenic plants and subsequent generations is of obvious academic and commercial importance. The isolation of promoters with a variety of expression profiles that are tissue-specific and/or temporally regulated will become more important as trait modification strategies evolve. Technologies such as site-directed integration, homologous recombination, ‘chimeraplasty’, and others will likely become routine in higher plants such as maize as this research area, now in its infancy, continues to develop. These technologies have the potential to aid our understanding of gene regulation, and to more directly make changes in endogenous gene sequences or to permit targeting of new genes (or regulatory elements) into precise genomic locations. With an assortment of accompanying genetic tools such as reverse genetic methods, mapping, genome-scale analysis and gene expression information, maize transformation has evolved into an important tool for both basic and applied studies in plants.

Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm

Cyclin-dependent kinases, the master regulators of the eukaryotic cell cycle, are complexes comprised of a catalytic serine/threonine protein kinase and an essential regulatory cyclin. The maize genome encodes over 50 cyclins grouped in different types, but they have been little investigated. We characterized a type B2 cyclin (CYCB2;2) during maize endosperm development, which comprises a cell proliferation phase based on the standard mitotic cell cycle, followed by an endoreduplication phase in which DNA replication is reiterated in the absence of mitosis or cytokinesis. CYCB2;2 RNA was present throughout the period of endosperm development studied, but its level declined as the endosperm transitioned from a mitotic to an endoreduplication cell cycle. However, the level of CYCB2;2 protein remained relatively constant during both stages of endosperm development. CYCB2;2 was recalcitrant to degradation by the 26S proteasome in endoreduplicating endosperm extracts, which could explain its sustained accumulation during endosperm development. In addition, although CYCB2;2 was generally localized to the nucleus of endosperm cells, a lower molecular weight form of the protein accumulated specifically in the cytosol of endoreduplicating endosperm cells. In dividing cells, CYCB2;2 appeared to be localized to the phragmoplast and may be involved in cytokinesis and cell wall formation. Kinase activity was associated with CYCB2;2 in mitotic endosperm, but was absent or greatly reduced in immature ear and endoreduplicating endosperm. CYCB2;2-associated kinase phosphorylated maize E2F1 and the “pocket” domains of RBR1 and RBR3. CYCB2;2 interacted with both maize CDKA;1 and CDKA;3 in insect cells. These results suggest CYCB2;2 functions primarily during the mitotic cell cycle, and they are discussed in the context of the roles of cyclins, CDKs and proteasome activity in the regulation of the cell cycle during endosperm development.