Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

RETINOBLASTOMA-RELATED protein stimulates cell differentiation in the Arabidopsis root meristem by interacting with cytokinin signaling.

Maintenance of mitotic cell clusters such as meristematic cells depends on their capacity to maintain the balance between cell division and cell differentiation necessary to control organ growth. In the Arabidopsis thaliana root meristem, the antagonistic interaction of two hormones, auxin and cytokinin, regulates this balance by positioning the transition zone, where mitotically active cells lose their capacity to divide and initiate their differentiation programs. In animals, a major regulator of both cell division and cell differentiation is the tumor suppressor protein RETINOBLASTOMA. Here, we show that similarly to its homolog in animal systems, the plant RETINOBLASTOMA-RELATED (RBR) protein regulates the differentiation of meristematic cells at the transition zone by allowing mRNA accumulation of AUXIN RESPONSE FACTOR19 (ARF19), a transcription factor involved in cell differentiation. We show that both RBR and the cytokinin-dependent transcription factor ARABIDOPSIS RESPONSE REGULATOR12 are required to activate the transcription of ARF19, which is involved in promoting cell differentiation and thus root growth.

The Lateral Root Cap Acts as an Auxin Sink that Controls Meristem Size.

Plant developmental plasticity relies on the activities of meristems, regions where stem cells continuously produce new cells [1]. The lateral root cap (LRC) is the outermost tissue of the root meristem [1], and it is known to play an important role during root development [2-6]. In particular, it has been shown that mechanical or genetic ablation of LRC cells affect meristem size [7, 8]; however, the molecular mechanisms involved are unknown. Root meristem size and, consequently, root growth depend on the position of the transition zone (TZ), a boundary that separates dividing from differentiating cells [9, 10]. The interaction of two phytohormones, cytokinin and auxin, is fundamental in controlling the position of the TZ [9, 10]. Cytokinin via the ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) control auxin distribution within the meristem, generating an instructive auxin minimum that positions the TZ [10]. We identify a cytokinin-dependent molecular mechanism that acts in the LRC to control the position of the TZ and meristem size. We show that auxin levels within the LRC cells depends on PIN-FORMED 5 (PIN5), a cytokinin-activated intracellular transporter that pumps auxin from the cytoplasm into the endoplasmic reticulum, and on irreversible auxin conjugation mediated by the IAA-amino synthase GRETCHEN HAGEN 3.17 (GH3.17). By titrating auxin in the LRC, the PIN5 and the GH3.17 genes control auxin levels in the entire root meristem. Overall, our results indicate that the LRC serves as an auxin sink that, under the control of cytokinin, regulates meristem size and root growth.

Spatial coordination between stem cell activity and cell differentiation in the root meristem.

A critical issue in development is the coordination of the activity of stem cell niches with differentiation of their progeny to ensure coherent organ growth. In the plant root, these processes take place at opposite ends of the meristem and must be coordinated with each other at a distance. Here, we show that in Arabidopsis, the gene SCR presides over this spatial coordination. In the organizing center of the root stem cell niche, SCR directly represses the expression of the cytokinin-response transcription factor ARR1, which promotes cell differentiation, controlling auxin production via the ASB1 gene and sustaining stem cell activity. This allows SCR to regulate, via auxin, the level of ARR1 expression in the transition zone where the stem cell progeny leaves the meristem, thus controlling the rate of differentiation. In this way, SCR simultaneously controls stem cell division and differentiation, ensuring coherent root growth.

Growth and development of the root apical meristem.

A key question in plant developmental biology is how cell division and cell differentiation are balanced to modulate organ growth and shape organ size. In recent years, several advances have been made in understanding how this balance is achieved during root development. In the Arabidopsis root meristem, stem cells in the apical region of the meristem self-renew and produce daughter cells that differentiate in the distal meristem transition zone. Several factors have been implicated in controlling the different functional zones of the root meristem to modulate root growth; among these, plant hormones have been shown to play a main role. In this review, we summarize recent findings regarding the role of hormone signaling and transcriptional networks in regulating root development.

Analysis of root meristem size development.

Plant post-embryonic development takes place in the meristems. In the root of the model plant Arabidopsis thaliana, stem cells organized in a stem-cell niche in the apex of the root meristem generate transit-amplifying cells, which undergo additional division in the proximal meristem and differentiate in the elongation/differentiation zone. For meristem maintenance, and therefore continuous root growth, the rate of cell differentiation must equal the rate of generation of new cells: how this balance is achieved is a central question in plant development. We have shown that maintenance of the Arabidopsis root meristem size is established by a balance between the antagonistic effects of cytokinin, which promotes cell differentiation, and auxin, which promotes cell division. Cytokinin antagonizes auxin in a specific developmental domain (the vascular tissue transition zone) from where it controls the differentiation rate of all the other root tissues. Here, we describe protocols to analyze development of root meristems.

The molecular basis of cytokinin function.

Cytokinins are a class of phytohormones that regulate a wide variety of physiological and developmental processes such as shoot and root growth. Cytokinin signaling relies on a phosphorelay mechanism similar to the prokaryotic two-component system. Although the principal components mediating this cascade have been identified, only recently have we begun to understand the molecular basis of cytokinin action. For example cytokinins control cell differentiation rate during root meristem development by suppressing both auxin signaling and transport, whereas at early stages of embryo development auxin counteracts cytokinin signaling to establish the embryonic root stem-cell niche. The antagonistic interaction between cytokinins and auxin seems to also occur in other developmental processes, such as lateral root emergence and leaf initiation.

The rate of cell differentiation controls the Arabidopsis root meristem growth phase.

Upon seed germination, apical meristems grow as cell division prevails over differentiation and reach their final size when division and differentiation reach a balance. In the Arabidopsis root meristem, this balance results from the interaction between cytokinin (promoting differentiation) and auxin (promoting division) through a regulatory circuit whereby the ARR1 cytokinin-responsive transcription factor activates the gene SHY2, which negatively regulates the PIN genes encoding auxin transport facilitators. However, it remains unknown how the final meristem size is set, i.e., how a change in the relative rates of cell division and differentiation is brought about to cause meristem growth to stop. Here, we show that during meristem growth, expression of SHY2 is driven by another cytokinin-response factor, ARR12, and that completion of growth is brought about by the upregulation of SHY2 caused by both ARR12 and ARR1: this leads to an increase in cell differentiation rate that balances it with division, thus setting root meristem size. We also show that gibberellins selectively repress expression of ARR1 at early stages of meristem development, and that the DELLA protein REPRESSOR OF GA 1-3 (RGA) mediates this negative control.

A genetic framework for the control of cell division and differentiation in the root meristem.

Plant growth and development are sustained by meristems. Meristem activity is controlled by auxin and cytokinin, two hormones whose interactions in determining a specific developmental output are still poorly understood. By means of a comprehensive genetic and molecular analysis in Arabidopsis, we show that a primary cytokinin-response transcription factor, ARR1, activates the gene SHY2/IAA3 (SHY2), a repressor of auxin signaling that negatively regulates the PIN auxin transport facilitator genes: thereby, cytokinin causes auxin redistribution, prompting cell differentiation. Conversely, auxin mediates degradation of the SHY2 protein, sustaining PIN activities and cell division. Thus, the cell differentiation and division balance necessary for controlling root meristem size and root growth is the result of the interaction between cytokinin and auxin through a simple regulatory circuit converging on the SHY2 gene.