Cell cycles and endocycles in the model brown seaweed Ectocarpus siliculosus

Abstract

The recent announcement of the first genome sequence of a brown macroalga, the filamentous Ectocarpus,¹ has been accompanied by a number of companion papers in New Phytologist. In a paper which contributes to this special issue, we classified the core cell cycle components of Ectocarpus, comparing them to the previously studied cell cycle components of diatoms. We then carried out fluorescence microscopy experiments to show that the Ectocarpus cell cycle could be deregulated during early development to give endopolyploid adults. We discuss here how our findings complement recent studies on endopolyploidy in plant and algal systems.

The brown macroalgae or seaweeds, remain a relatively poorly understood eukaryotic clade, despite their considerable evolutionary² and ecological importance.³ Their developmental biology, in particular, is of fundamental interest, as it helps to shape the structure of Earth's coastal ecosystems. The recent completion of the first brown macroalgal genome sequence,¹ that of the model organism⁴ Ectocarpus (Fig. 1), affords new insights into the developmental processes of brown seaweeds.

Figure 1.

Upright filaments of a mature Ectocarpus sporophyte. U, unilocular sporangia, which produce spores through meiosis (or through apomeiosis in some partheno-sporophytes²³); P, plurilocular sporangia which produce spores through mitosis.

Like many of the brown seaweeds, Ectocarpus displays a remarkable diversity of developmental forms. For simplicity's sake, its basic life cycle is commonly presented as the sexual alternation of a haploid gametophyte and a diploid sporophyte, both of which consist of branched multicellular filaments (Fig. 1). However, this textbook alternation of generations is overlaid by a broad range of developmental pathways in which generations are only loosely connected by sexuality and only loosely coupled to ploidy.⁵ This complexity has been aptly described as a Netzwerk von Entwicklungsmöglichkeiten—a ‘network of developmental possibilities’—in which ‘all observed nuclear ploidies and generations are connected with each other by meiosis, heteroblasty, gamete fusion and endoreduplication’.⁶

Such a level of developmental plasticity requires flexible co-ordination⁷ of DNA replication, cell division and cell morphogenesis, especially during embryogenesis and early development when the body axes of the mature Ectocarpus plants are specified. At its most extreme, this can lead to cell cycle deregulation and endopolyploidy, in which the replication of nuclear DNA is completely uncoupled from concomitant cell division, leading to a heritable increase in chromosome number. Although its functional significance is often debated,⁸ endopolyploidy is impressively common in plants⁹ and animals,¹⁰ and is frequently associated with, and often assumed to drive, normal^11,12 and abnormal¹³ growth, development and differentiation. In leaves of the flowering plant, Arabidopsis thaliana, for example, several rounds of endoreduplication occur in trichomes and in most epidermal pavement cells, while guard cells remain diploid, with recent work confirming the importance of endopolyploidy in directing cell fate in these leaves.¹⁴

Regardless of its functional significance, endopolyploidy represents, mechanistically, an uncoupling of the normal cell cycle and theory and practice have both shown that this deregulation is centred on the Cyclin Dependent Kinase (CDK)-cyclin complexes. CDK-cyclin complexes are the engines of eukaryotic cell cycle progression, with expression and degradation of specific cyclins promoting progression through and exit from, distinct stages of the cell cycle. In plants, CDKA activity following S-phase DNA replication drives the entry into mitosis (Fig. 2A), and if this activity is suppressed, the cell will not divide after DNA replication, resulting in endopolyploidy. This CDK-cyclin suppression may happen in any of three ways¹⁵ (Fig. 2B). First, the CDKA-cyclin complex may be inhibited through phosphorylation by the Wee1 protein kinase. Experiments in tomato fruit, in which endopolyploidy is associated with fruit growth,¹⁶ have shown that underexpression of this kinase leads to a decrease in cell ploidy levels.¹⁷ Second, the cyclin partners of the CDKA-cyclin complex may be marked for proteolysis by the concerted action of CCS52 proteins and the anaphase-promoting complex, an E3 ubiquitin ligase. Again, underexpression of CCS52 in tomato fruits has been shown to cause a reduction in the levels of endopolyploidy.¹⁸ Third and finally, CDKA-cyclin activity may be moderated by the binding of regulatory proteins, such as those of the RBR/E2F/DP/DEL pathway and the cyclin-dependent kinase inhibitors (CKI). The major class of CKIs in seed plants are the Kip-Related Proteins or KRPs and, as before, underexpression of these reduces ploidy levels in tomato.¹⁹

Figure 2.

Cell cycle regulatory networks differ in the mitotic and endoreduplicative cell cycles. Overview (after Inzé and De Veylder 2006) of the proteins thought to be involved in regulating (a) the mitotic cell cycle and (b) the endocycle in green plants. Symbols with black lettering indicate activated proteins, grey symbols with white lettering indicate proteins whose activity is inhibited. High CD KA/Cyc and CD KB/Cyc activities drive mitosis and are maintained by CD KB/CycA/B—which is under transcriptional control by the E2F pathway—inhibition of KRP proteins and by CD KD/CycH activity. Entry into the endocycle is associated with higher levels of KRP activity and CC S52 activity, leading to CD KA/Cyc and CD KB/Cyc inhibition.

Despite this large body of work on endopolyploidy in seed plants, little is known about its prevalence or mechanism in the brown seaweeds. Previous studies have reported endopolyploidy in brown seaweed species^6,20,21 but, despite a large body of physiological and cell biological work on the brown algal embryonic cell cycle, especially in Fucoid species,^7,22 few cell cycle or endocycle components have been identified.

Accordingly, in a recent paper,²³ we took advantage of the recent release of the Ectocarpus genome sequence¹ to take a first look at the molecular components of the brown seaweed cell cycle, carrying out further experiments to study the way in which endopolyploidy contributes to the Ectocarpus life cycle.

The Ectocarpus genome was first searched for core cell cycle genes:^24–27 CDKs and cyclins, together with those regulators of mitotic CDK-cyclin complexes which have been shown to allow plants to switch between normal cell cycle and endopolyploidy.¹⁵ We showed that Ectocarpus has a relatively concise complement of cell cycle genes and, interestingly, lacks any obvious homologs to the plant KRP CDK-cyclin inhibitors.²³ The characterisation of this cell cycle regulatory network will lay the foundation for future gene expression and manipulation studies which will show how this network is organized and controlled during development.

After this, single-cell microscopy studies were carried out to elucidate how endopolyploidy fits into the Ectocarpus life cycle. Previous chromosome counting studies, which we confirmed using flow cytometry of Ectocarpus populations, had shown that parthenogenetically-derived sporophytes, which had developed from haploid (C) gametes, could contain 2C nuclei.⁶ However, this technique relied upon finding cells in metaphase and so could only identify the ploidy of one or two cells in each individual. We therefore used Hoechst 33342, a fluorescent DNA-binding stain, to measure the ploidy of individual cells during the first two weeks of Ectocarpus development. Our results showed that the first cell cycle in Ectocarpus may be mitotic or endoreduplicative, and that the ploidy of adult parthenosporophytes depends on whether or not endoreduplication occurs during their first cell division. Thus, all the nuclei in any given parthenosporophyte have the same ploidy, and there is no obvious correlation between endopolyploidy and cell size or cell fate. This is unusual, because endopolyploidy in most eukaryotes is believed to occur during late development, leading to the sort of mosaic endopolyploidy that has been reported in the kelps Laminaria saccharina and Alaria esculenta.²¹ However, the extent to which endopolyploidy is related to cell fate in brown seaweeds may depend upon the extent of cellular differentiation in different seaweed species; in Ectocarpus, there is only a limited range of cell types and all cells are roughly the same size. In the kelps L. saccharina and A. esculenta, on the other hand, there is a wider range of cell types and sizes in any given plant and, although the association between cell size and cell ploidy is not absolute, it is likely that mosaic development may reflect this wider range of cell types.²¹

A final, alternative hypothesis is based on the fact that endopolyploidy occurs in a broad variety of arthropods, mammals and plants which, like Ectocarpus, have a small genome and a short life cycle.^9,28 This may be because diploid development provides gene redundancy, buffers epigenetic defects, allows double strand breaks to be repaired by homologous and nonhomologous recombination,²⁹ and may allow an optimal balance between organellar and nuclear DNA levels, enhancing metabolic capacity. Such protective effects may be important as Ectocarpus may live in the intertidal zone and is exposed to regular and multiple biotic and abiotic stresses. While the functional significance of endopolyploidy thus remains unclear, there are promising lines of enquiry which remain to be investigated and we hope that our research serves to stimulate these.

Addendum to: Bothwell JH, Marie D, Peters AF, Cock JM, Coelho SM. Role of endoreduplication and apomeiosis during parthenogenetic reproduction in the model brown alga Ectocarpus. New Phytol. 2010;188:111–121. doi: 10.1111/j.1469-8137.2010.03357.x.