Tuning the cell-cycle engine for improved plant performance Gerrit TS Beemster, Vladimir Mironov and Dirk Inze´ Cell-cycle regulation plays a crucial role in organogenesis, morphogenesis, growth and differentiation and conceptually offers a means to design a next generation of crop plants that outperform traditionally bred ones. However, cell-cycle regulation involves a large, highly redundant, set of genes, which complicates unravelling of function in the context of a higher plant. Nevertheless, ten years of molecular cell-cycle research, primarily in the model plant Arabidopsis, have demonstrated its potential for altering plant development. Addresses Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB)/Ghent University, Technologiepark 927, Ghent, Belgium Corresponding author: Inze´, Dirk (dirk.inze@psb.ugent.be)
Current Opinion in Biotechnology 2005, 16:142–146 This review comes from a themed issue on Plant biotechnology Edited by Dirk Inze´ Available online 11th February 2005 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.01.006
Introduction Early microscopists concluded that cells are the building blocks of higher plants. They postulated that cell division, by influencing the number of cells per organ, and cell expansion, by influencing their final size, together determine organ growth rate. Although the interrelationship between expansion and division is poorly understood [1,2], this framework has proven useful. Cell number and size differences both contribute to variations in leaf size in response to environmental conditions [3–7], genetic [8–10] and age-dependent differences [11]. Although in some exceptional cases (e.g. Characean algae) single cells can reach some centimetres in length [12], in general, the importance of the cell division process becomes evident from comparisons of leaf area between species: that of Arabidopsis thaliana (16 mm2) is tiny compared with those of sunflower (18 000–30 000 mm2), yet epidermal cell size is remarkably similar (1200 mm2 and 1500 mm2, respectively [4,13]). Similarly, epidermal cell length in leaves from Poa alpina ( 41 mm long) nearly equals that in corn leaves (up to 1 m in length): 120 mm versus 150 mm [8,14]. Current Opinion in Biotechnology 2005, 16:142–146
Nevertheless, differences in mature cell size should not be discarded as a means of increasing crop productivity. Polyploid varieties of various crop species grow larger than diploid varieties [15]. Furthermore, many diploid plants contain polyploŨd cells [16] as a result of endoreduplication, a cell cycle consisting of successive rounds of DNA duplication in the absence of mitosis. Differences in nuclear DNA content often correlate closely with cell size [17,18]. Moreover, endoreduplication co-occurs with cell expansion, suggesting a functional relationship [19,20 ]. The current consensus, based on analyses of endopolyploidy and systemic or clonal polyploids, is that ploŨdy determines the growth potential, whereas its realization depends on the context [21,22].
The cell-cycle engine of growth To understand the relationship between cell-cycle activity and growth, we need to analyse growth from a cellular perspective. Cell-cycle activity occurs in growth zones that encompass a meristem where cells are proliferating and a zone where cells expand to mature size. Different types of growth zones in higher plants are variations on this theme (Figure 1), implying common principles of (growth) regulation. Two regulatory principles can be distinguished: progression from one stage to the next (Figure 1; grey arrows) and the rate of the growth process (Figure 1; white arrows). For the shoot apical meristem, mutant analysis unearthed the regulatory system that controls the transition of cells from one stage to the next [23]. The importance of meristem size in the control of organ growth rates (Figure 1; 7) was demonstrated in the root of Arabidopsis, where growth rate was proportional to meristem size [24]. The rate of proliferation (Figure 1; 3) is inversely proportional to cell-cycle duration (Tc). Conceptually, differences in average cell-cycle duration can be brought about in two ways: equal variations of Tc in all cells and variations in the fraction of cells that divide. Experimental evidence suggested the involvement of variations in the proliferative fraction in growth regulation [25,26]. However, the techniques employed were designed for cell cultures, and might lead to artefacts in plant organs. Experimental approaches designed for in planta analyses suggest a uniform cell-cycle duration throughout the meristem [27]. Owing to proliferative activity, cells are continuously displaced out of the meristem into the adjacent expansion zone. In this region, cells expand to many times their original size. Effectively, expansion functions as a ‘gearbox’ between the ‘cell-cycle engine’ and organ growth. www.sciencedirect.com
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Figure 1
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Regulatory mechanisms employed in the growth zones of plants. In most plants, growth zones comprise four stages separated in time or space: quiescence, initiation, active proliferation and endoreduplication/differentiation. Cells in these stages are adjacent to the mature tissue that makes up the bulk of the plant. Regulation of the growth zone as a whole occurs at two levels: regulation of the rate at which growth processes progress (e.g. regulation of the cell cycle and cell growth; arrows 1–4), and regulation of the transition from one stage to the next (arrows 5–8).
The expansion of cells depends on expansion rate (Figure 1; 4) and the size of the expansion zone (Figure 1; 8). Both of these parameters modulate growth in response to experimental treatments [24,28,29].
The cell-cycle regulatory mechanism The study of cell-cycle control in plants ensued with the discovery in the late 1980s of a conserved machinery homologous to that of yeasts and mammals. Subsequently, plant cell-cycle genes were cloned, and a complete list was composed for the model species A. thaliana [30 ]. The conserved nature of the cell-cycle ‘engine’ implies that the same principles and molecules are involved in all eukaryotes, but in terms of function it is impossible to match genes from evolutionarily distant organisms in a one-to-one manner. For example, plants do not have obvious homologues of some key cell-cycle regulators, such as E-type cyclins or CDC25, whereas B-type cyclin-dependent kinases (CDKs) appear to be plant-specific. The diversity of other types of molecules in plants (e.g. cyclins A and B) is bewildering; therefore, the function of all these cell-cycle regulators needs to be elucidated in planta.
Evidence from transgenic plants The most conclusive way to establish gene function is to study the effects of altered expression. Many such studies have been performed over the past decade and it is timely to review the findings. Effects on cell production
The first experiments with transgenic plants in which the activity of the A-type CDK gene CDKA;1 was altered were carried out 10 years ago. Although increasing its levels had no effect on development, inhibiting its activity by expressing a dominant negative mutant of the protein inhibited cell proliferation in tobacco. However, the effect on cell division was compensated for, albeit not www.sciencedirect.com
completely, by the formation of larger cells [31]. This observation was reminiscent of classical experiments with wheat seedlings from gamma-irradiated seeds, which lack cell proliferation [32]. Although the growth of these seedlings was slower, and even ceased completely at day 12 after sowing, this paper is often cited as showing the independence of growth on cell division. In line with its function as a CDK inhibitor, the cell-cycle inhibitory protein KRP2 induced a strong reduction in CDK activity and cell-division rates in Arabidopsis. Despite cell size compensation the plants were dwarfed and leaves became serrated [13]. It was shown that the growth reduction was primarily the result of a lower division rate of meristematic cells (Figure 1; 3). The increased cell size originated from the meristem and was maintained in the absence of an effect on the expansion process. Inhibition of the cell cycle in meristematic cells is not always compensated for by the formation of larger cells. Overexpression of the CDK subunit CKS1 inhibited cellcycle progression specifically in the meristem and reduced growth without compensatory changes in the cell size [33]. Similarly, no compensation was observed when cell production was strongly attenuated in petals of Brassica napus overproducing KRP1 [34]. Inversely, when cell proliferation was stimulated by enhanced expression of the mitotic CYCB1;1, root growth rates were significantly increased in the absence of an effect on cell size [35]. Similarly, constitutive expression of CYCB2;2 in rice plants resulted in accelerated root growth [36]. Elevated expression of the Arabidopsis S-phase cyclin CYCD2;1 in tobacco also resulted in higher growth rates and accelerated development, but had no effect on the cell volume or final organ size [37]. With the exception of the CKS1overexpressing plants, unfortunately, none of these transgenics was characterized in terms of the effects on growth zone parameters. Current Opinion in Biotechnology 2005, 16:142–146
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Effects on the switch between proliferation and differentiation
Assuming that transitions from proliferation to differentiation (Figure 1; 7) and differentiation to maturity (Figure 1; 8) are independent, delaying the former should lead to small organs consisting of huge numbers of undifferentiated cells. Indeed, overexpression of CYCD3;1 led to hyperproliferation in Arabidopsis leaves, with large numbers of small, undifferentiated cells in which endoreduplication was inhibited [38]. del Pozo et al. [39] showed that overexpression of a stable form of the transcription factor E2Fc, which is normally expressed in differentiating cells, inhibits cell division and increases cell size. When CYCB1;2 was overexpressed in Arabidopsis trichomes (hair cells that normally undergo extensive endoreduplication), a strong reduction in ploidy was accompanied by a proportional induction of division in the trichomes, without changes in the total number of cell cycles or trichome size [40]. Similarly, downregulation of CCS52, a negative regulator of mitotic cyclins in Medicago sativa, increased the number of divisions at the expense of endocycles without an impact on organ growth [41]. Effects on endoreduplication only
In addition to having an effect on cell division, ectopic expression of KRP1 in Arabidopsis trichomes reduced ploidy level without changes in cell number and strongly reduced trichome size and number of branches [42]. Constitutive overexpression of DEL1, which is normally only expressed in proliferating cells, specifically inhibited endoreduplication (Figure 1; 4) and decreased mature cell size in Arabidopsis leaves, whereas its downregulation had the opposite effect [43]. Another member of the DEL family, E2Ff, specifically suppresses the expression of cell expansion genes during the proliferation phase, releasing them in the expansion phase [44 ]; thus, it seems that the function of this gene family is to suppress differentiation processes in proliferating tissues. Overexpression of the putative DNA replication activator CDC6, a target of E2F, boosted endoreduplication in Arabidopsis [45], but unfortunately no phenotypic description of the transgenic plants was provided. Effects on proliferation and endoreduplication
Like overproduction of their upstream regulator CYCD3;1, simultaneous constitutive expression of both the transcription facor E2Fa and its dimerization partner DPa resulted in overproliferation. However, in this case cells that lost their mitotic potential underwent supernumerary rounds of endoreduplication, demonstrating the involvement of the E2F–DP complex in both mitotic and endocycle regulation [46,47] (Figure 1; 3 and 4). Similarly, when CYCD3;1 was overproduced in trichomes it enhanced divisions and induced supernumerary endoreduplication, considerably increasing trichome size and Current Opinion in Biotechnology 2005, 16:142–146
number of trichomes, per trichome initiation site [48 ]. This observation demonstrates that the same gene can have multiple effects in different tissues. Surprises
Although the previous examples fit within the framework depicted in Figure 1, surprising results were obtained when cell proliferation was manipulated locally in tobacco meristems or leaf promordia. The induction of CYCA3;2 or the yeast CDC25 phosphatase caused a local and transient increase in cell division [49 ]. In the meristem this led to leaf initiation, whereas the morphology of the formed leaves was unaffected. In developing leaf primordia, the same genes promoted a transient increase in cell division rates; however, the effect on lamina development seen after the leaf matured was an indentation of the affected region. In other words, a transient increase in cell division inhibited overall growth. Conversely, the application to leaf promordia of the CDK-inhibitory compound roscovitin inhibited division and caused overgrowth of the affected part of the lamina. These results demonstrate that the role of cell-cycle progression is context-dependent and sometimes unpredictable.
Conclusions The studies presented here demonstrate the enormous and versatile potential of the cell-cycle regulatory system to alter plant growth. Here, we have fitted published results obtained from studies of the effect of the misexpression of cell-cycle genes in a functional framework to enable better understanding of the relationship between cell-cycle changes and the effect on overall organ growth. Unfortunately, detailed analyses of the effects on the dynamics of all cell-cycle processes, rather than a description of the mature stage, are still an exception, making this task difficult and to some extent speculative. Powerful methods to perform such analyses have been developed and should be applied to fully understand the relationship between cell-cycle regulation and organ growth [20 ]. To use cell-cycle genes to improve crop growth characteristics we will need to understand differences in the effect of related genes to enable prediction of the effect of a particular perturbation. To achieve this it will be necessary to start analysing the cell cycle as a system of interacting components, shifting away from a gene-by-gene description of the phenotype. This may be challenging, but will ultimately be the only way to effectively manage the raw potential encompassed by this regulatory mechanism.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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