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.

Cell cycle control and seed development.

Seed development is a complex process that requires coordinated integration of many genetic, metabolic, and physiological pathways and environmental cues. Different cell cycle types, such as asymmetric cell division, acytokinetic mitosis, mitotic cell division, and endoreduplication, frequently occur in sequential yet overlapping manner during the development of the embryo and the endosperm, seed structures that are both products of double fertilization. Asymmetric cell divisions in the embryo generate polarized daughter cells with different cell fates. While nuclear and cell division cycles play a key role in determining final seed cell numbers, endoreduplication is often associated with processes such as cell enlargement and accumulation of storage metabolites that underlie cell differentiation and growth of the different seed compartments. This review focuses on recent advances in our understanding of different cell cycle mechanisms operating during seed development and their impact on the growth, development, and function of seed tissues. Particularly, the roles of core cell cycle regulators, such as cyclin-dependent-kinases and their inhibitors, the Retinoblastoma-Related/E2F pathway and the proteasome-ubiquitin system, are discussed in the contexts of different cell cycle types that characterize seed development. The contributions of nuclear and cellular proliferative cycles and endoreduplication to cereal endosperm development are also discussed.

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization.

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin-proteasome pathway.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm.

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

The contribution of cell cycle regulation to endosperm development.

Development of the seed endosperm involves several different types of coordinated cell cycle programs: acytokinetic mitosis, which produces a syncytium soon after fertilization; cellularization through the formation of modified phragmoplasts; cell proliferation, in which mitosis is coupled to cell division; and, in certain species like cereal crops, endoreduplication. Understanding the regulation of these programs and their transitions is challenging, but it has the potential to define important links between the cell cycle, cell differentiation and development, as well as provide tools for the manipulation of seed yield. A relatively large number of mutants display endosperm proliferation defects, and connections with known cell cycle genes are beginning to emerge. For example, it is becoming increasingly evident that the master cell cycle regulators, the cyclin-dependent kinases and retinoblastoma-related families, play key roles in the events leading to endosperm formation and development. Recent studies highlight cross-talk between pathways controlling the cell cycle and genomic imprinting.

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.

Grasses like mammals? Redundancy and compensatory regulation within the retinoblastoma protein family.

The retinoblastoma (RB) protein family plays a conserved and inhibitory role in cell cycle progression in higher eukaryotes. In mammals, this family includes, in addition to RB, the related (RBR) proteins p107 and p130, which appear to have both specific and redundant functions compared to those of the prototypical RB protein. Whereas most plant species seem to possess only one RBR gene, a recent study has shown that in maize there are two types of distinctly regulated RBR proteins, RBR1 and RBR3. Expression of RBR3 RNA is controlled by the RBR1-E2F pathway, and it is upregulated upon inhibition of RBR1 activity by the wheat dwarf virus RepA protein in tissue culture, indicating the presence of a specific compensatory mechanism sustaining high pocket protein activity. Database mining and phylogenetic analyses suggest the presence of two distinct RBR genes to be a unique feature of grasses among plants, which might help to explain their recalcitrance to genetic transformation.

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.

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 p13(Suc1). 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.

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 gamma-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.