MOLECULAR REGULATORS OF PLANT GROWTH

Plants and animals have different developmental strategies for growth, yet individuals of both attain characteristic species-specific sizes constrained by their developmental genetic programmes. Additionally, growth can be significantly influenced by environmental factors, specifically so in plants. We are seeking to understand how plant growth is regulated at the molecular level and through which molecular mechanisms environmental signals are able to influence growth.

The key factor in growth is the duration of cell proliferation and the timing of the exit from proliferation to cell expansion and differentiation (Figure 1). In plants, cell proliferation is largely concentrated in specialised regions known as meristems, which contain the stem cells. In meristems udifferentiated cells are produced by cell proliferation, and when these cells stop dividing, as they leave the meristematic region they differentiate into specific tissues. During differentiation, plant cells frequently increase their DNA content by a modified mitotic cycle called endoreduplication, a process of continous DNA synthesis without intervening mitosis. We are interested in the molecular mechanisms which maintain stem cell activity in the meristems; control the balance between cell division and differentiation and regulate the switch from mitotic cell cycle to endoreduplication in a parallel fashion during organ development (Figure 1). Our main interest is in genes involved in the regulatory mechanisms which make the decision to enter or leave the division cycle. We cloned a family of genes from the model plant Arabidopsis thaliana called E2F transcription factors, which are related to genes that control the same process in animals. The canonical role for E2F transcription factors is to regulate cell cycle entry, but it is becoming apparent in many sytems that E2Fs have broader functions and that, besides the regulation of cell cycle transitions, they also coordinate cell proliferation with cell growth and differentiation. According to the current model, E2Fs can work both as positive and negative regulators of transcription, depending on their structure and on the function of the retinoblastoma (RB) tumour suppressor protein.

Auxin is a plant growth hormone that regulates cell division in a concentration dependent manner; elevated auxin levels activate cell division in the meristems, whereas reduced amounts repress mitosis as cells leave the meristematic regions, and in parallel it enhances cell growth. We discovered the auxin increases the stability of the E2FB protein, and co-expression of E2FB with its dimerization partner DPA in plant cells could maintain cell proliferation in the absence of auxin. Cytokinin, another plant hormone works opposite to auxin and the antagonistic functions of these two hormones appear to be a key mechanism which regulates meristematic functions. Our recent work indicates that cytokinin can change the activity of E2FB from a transcriptional activator to a transcriptional repressor. Our aim is to understand the molecular mechanisms leading to this switch in E2F activity during hormone signalling, and to identify the downstream targets of E2Fs by using the chromatin immunoprecipitation (ChIP) method.

Figure 1. Mechanisms for organ size control. (a) Organ formation, exemplified here by leaf development, consist of two stages. The first phase is underpinned by cell proliferation, characterized by intense macromolecular/cytoplasmic synthesis and rapid cell division. The second phase is characterized by cell expansion and differentiation. Differentiation takes place along a basipetal gradient (that is, from leaf tip to leaf base), as indicated here by the gradient in cell size and cell greening. The red arrow summarizes proliferative inputs, and the black arrow indicates the arrest of proliferation and the initiation of differentiation. (b,c) The two principal mechanisms for controlling organ size. Enlargement of organs can be produced by either (b) increasing proliferation signals or (c) delaying the transition between proliferation and differentiation. In both cases the number of cells available for organ formation at the end of the proliferative phase is increased, but the underlying mechanisms are different (Bögre et al., Genome Biology 2008)

The maintenance of stem cells in the plant meristems is crucial for the growing plant. Previous studies demonstrated that the retinoblastoma-related protein 1 (RBR1) is a stem cell regulator in plants. Recently we have found that ectopic co-expression of E2FB and DPA heterodimeric transcription factors increases the amount of stem cells in Arabidopsis roots (Figure 2), which further supports the involvement of the plant RBR-E2F pathway in the regulation of stem cell maintenance.

Figure 2. Ectopic co-expression of E2FB with DPA increases the amount of stem cells in the root meristem of Arabidopsis as indicated by red arrow. Position of the quiescent centre (QC) is indicated.

To unravel the molecular pathway controlling the switch from proliferation to differentiation, we use the first developing leaf pair of Arabidopsis thaliana as a model system. In this model system, cells gradually exit the mitotic cell cycle and engage into an endoreduplication cycle as they start to differentiate (Figure 1). Our results indicate that different E2F proteins enter into complex with the single RBR1 protein at different developmental stages. We suggest that two E2Fs from Arabidopsis, E2FA and E2FB have antagonistic functions when they form complexes with RBR1; E2FA-RBR1 keeps cells in the mitotic cycle, whereas binding of RBR1 to E2FB stimulates cell cycle exit during leaf development (Figure 3). Our major aim is to identify the downstream targets of these E2F transcriptional complexes. Stress such as drought could change the activity of E2F complexes, which might lead to growth repression. We seek to understand how and why under stress conditions plants stop growing, and what happens at the cellular level. Molecular insights into this process and how it is signalled may lead to opportunities to engineer crops with increased stress tolerance and, consequently, with higher yields.

Figure 3. Proposed working model for E2FA and E2FB function when they form complexes with RBR1.