Gerrit T.S. Beemster

Functional Analysis of Cyclin-Dependent Kinase Inhibitors of Arabidopsis

Cyclin-dependent kinase inhibitors, such as the mammalian p27Kip1 protein, regulate correct cell cycle progression and the integration of developmental signals with the core cell cycle machinery. These inhibitors have been described in plants, but their function remains unresolved. We have isolated seven genes from Arabidopsis that encode proteins with distant sequence homology with p27Kip1, designated Kip-related proteins (KRPs). The KRPs were characterized by their domain organization and transcript profiles. With the exception of KRP5, all presented the same cyclin-dependent kinase binding specificity. When overproduced, KRP2 dramatically inhibited cell cycle progression in leaf primordia cells without affecting the temporal pattern of cell division and differentiation. Mature transgenic leaves were serrated and consisted of enlarged cells. Although the ploidy levels in young leaves were unaffected, endoreduplication was suppressed in older leaves. We conclude that KRP2 exerts a plant growth inhibitory activity by reducing cell proliferation in leaves, but, in contrast to its mammalian counterparts, it may not control the timing of cell cycle exit and differentiation.

Ethylene Upregulates Auxin Biosynthesis in Arabidopsis Seedlings to Enhance Inhibition of Root Cell Elongation

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion.

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal.

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.

Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor.

New plant cells arise at the meristems, where they divide a few times before they leave the cell‐cycle program and start to differentiate. Here we show that the E2Fa–DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co‐expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa–DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa–DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development.

Cell Cycle Progression in the Pericycle Is Not Sufficient for SOLITARY ROOT/IAA14-Mediated Lateral Root Initiation in Arabidopsis thaliana

To study the mechanisms behind auxin-induced cell division, lateral root initiation was used as a model system. By means of microarray analysis, genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the auxin/indole-3-acetic acid (AUX/IAA) signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation.

Gibberellin Signaling in the Endodermis Controls Arabidopsis Root Meristem Size

Plant growth is driven by cell proliferation and elongation. The hormone gibberellin (GA) regulates Arabidopsis root growth by controlling cell elongation, but it is currently unknown whether GA also controls root cell proliferation. Here we show that GA biosynthetic mutants are unable to increase their cell production rate and meristem size after germination. GA signals the degradation of the DELLA growth repressor proteins GAI and RGA, promoting root cell production. Targeting the expression of gai (a non-GA-degradable mutant form of GAI) in the root meristem disrupts cell proliferation. Moreover, expressing gai in dividing endodermal cells was sufficient to block root meristem enlargement. We report a novel function for GA regulating cell proliferation where this signal acts by removing DELLA in a subset of, rather than all, meristem cells. We suggest that the GA-regulated rate of expansion of dividing endodermal cells dictates the equivalent rate in other root tissues. Cells must double in size prior to dividing but cannot do so independently, because they are physically restrained by adjacent tissues with which they share cell walls. Our study highlights the importance of probing regulatory mechanisms linking molecular- and cellular-scale processes with tissue and organ growth responses.

Gibberellin Signaling Controls Cell Proliferation Rate in Arabidopsis

Plant growth involves the integration of many environmental and endogenous signals that together with the intrinsic genetic program determine plant size. At the cellular level, growth rate is regulated by the combined activity of two processes: cell proliferation and expansion. Gibberellins (GA) are plant-specific hormones that play a central role in the regulation of growth and development with respect to environmental variability. It is well established that GA promote growth through cell expansion by stimulating the destruction of growth-repressing DELLA proteins (DELLAs); however, their effects on cell proliferation remain unknown. Kinematic analysis of leaf and root meristem growth revealed a novel function of DELLAs in restraining cell production. Moreover, by visualizing the cell cycle marker cyclinB1::beta-glucuronidase in GA-signaling mutants, we show that GA modulate cell cycle activity in the root meristem via a DELLA-dependent mechanism. Accordingly, expressing gai (a nondegradable DELLA protein) solely in root meristem reduced substantially the number of dividing cells. We also show that DELLAs restrain cell production by enhancing the levels of the cell cycle inhibitors Kip-related protein 2 (KRP2) and SIAMESE (SIM). Therefore, DELLAs exert a general plant growth inhibitory activity by reducing both cell proliferation and expansion rates, enabling phenotypic plasticity.

Genome-Wide Analysis of Gene Expression Profiles Associated with Cell Cycle Transitions in Growing Organs of Arabidopsis

Organ growth results from the progression of component cells through subsequent phases of proliferation and expansion before reaching maturity. We combined kinematic analysis, flowcytometry, and microarray analysis to characterize cell cycle regulation during the growth process of leaves 1 and 2 of Arabidopsis (Arabidopsis thaliana). Kinematic analysis showed that the epidermis proliferates until day 12; thereafter, cells expand until day 19 when leaves reach maturity. Flowcytometry revealed that endoreduplication occurs from the time cell division rates decline until the end of cell expansion. Analysis of 10 time points with a 6k-cDNA microarray showed that transitions between the growth stages were closely reflected in the mRNA expression data. Subsequent genome-wide microarray analysis on the three main stages allowed us to categorize known cell cycle genes into three major classes: constitutively expressed, proliferative, and inhibitory. Comparison with published expression data obtained from root zones corresponding to similar developmental stages and from synchronized cell cultures supported this categorization and enabled us to identify a high confidence set of 131 proliferation genes. Most of those had an M phase-dependent expression pattern and, in addition to many known cell cycle-related genes, there were at least 90 that were unknown or previously not associated with proliferation.

The Plant-Specific Cyclin-Dependent Kinase CDKB1;1 and Transcription Factor E2Fa-DPa Control the Balance of Mitotically Dividing and Endoreduplicating Cells in Arabidopsis

Transgenic Arabidopsis thaliana plants overproducing the E2Fa-DPa transcription factor have two distinct cell-specific phenotypes: some cells divide ectopically and others are stimulated to endocycle. The decision of cells to undergo extra mitotic divisions has been postulated to depend on the presence of a mitosis-inducing factor (MIF). Plants possess a unique class of cyclin-dependent kinases (CDKs; B-type) for which no ortholog is found in other kingdoms. The peak of CDKB1;1 activity around the G2-M boundary suggested that it might be part of the MIF. Plants that overexpressed a dominant negative allele of CDKB1;1 underwent enhanced endoreduplication, demonstrating that CDKB1;1 activity was required to inhibit the endocycle. Moreover, when the mutant CDKB1;1 allele was overexpressed in an E2Fa-DPa–overproducing background, it enhanced the endoreduplication phenotype, whereas the extra mitotic cell divisions normally induced by E2Fa-DPa were repressed. Surprisingly, CDKB1;1 transcription was controlled by the E2F pathway, as shown by its upregulation in E2Fa-DPa–overproducing plants and mutational analysis of the E2F binding site in the CDKB1;1 promoter. These findings illustrate a cross talking mechanism between the G1-S and G2-M transition points.

Cell cycle: the key to plant growth control?

Experimental evidence on the role of the cell cycle in plant growth regulation does not exclusively fit the cellular (division drives growth) or the organismal perspective (division merely accompanies growth). Here we present a broader, integrated concept of plant growth regulatory interactions, which accommodates experimental results gathered to date. This model can serve as a basis for future research, and prompts experimental approaches to encompass both measurements of cell growth and division parameters.