Plant and animal stem cells: similar yet different.

The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into new tissues. Plant stem cell niches are located within the meristems, which are organized structures that are responsible for most post-embryonic development. The continuous organ production that is characteristic of plant growth requires a robust regulatory network to keep the balance between pluripotent stem cells and differentiating progeny. Components of this network have now been elucidated and provide a unique opportunity for comparing strategies that were developed in the animal and plant kingdoms, which underlie the logic of stem cell behaviour.

The Mutual Inhibition between PLETHORAs and ARABIDOPSIS RESPONSE REGULATORs Controls Root Zonation.

During organogenesis, a key step toward the development of a functional organ is the separation of cells into specific domains with different activities. Mutual inhibition of gene expression has been shown to be sufficient to establish and maintain these domains during organogenesis in several multicellular organisms. Here, we show that the mutual inhibition between the PLETHORA transcription factors (PLTs) and the ARABIDOPSIS RESPONSE REGULATORs (ARRs) transcription factors is sufficient to separate cell division and cell differentiation during root organogenesis. In particular, we show that ARR1 suppresses PLT activities and that PLTs suppress ARR1 and ARR12 by targeting their proteins for degradation via the KISS ME DEADLY 2 F-box protein. These findings reveal new important aspects of the complex process of root zonation and development.

Acidic cell elongation drives cell differentiation in the Arabidopsis root.

In multicellular systems, the control of cell size is fundamental in regulating the development and growth of the different organs and of the whole organism. In most systems, major changes in cell size can be observed during differentiation processes where cells change their volume to adapt their shape to their final function. How relevant changes in cell volume are in driving the differentiation program is a long-standing fundamental question in developmental biology. In the Arabidopsis root meristem, characteristic changes in the size of the distal meristematic cells identify cells that initiated the differentiation program. Here, we show that changes in cell size are essential for the initial steps of cell differentiation and that these changes depend on the concomitant activation by the plant hormone cytokinin of the EXPAs proteins and the AHA1 and AHA2 proton pumps. These findings identify a growth module that builds on a synergy between cytokinin-dependent pH modification and wall remodeling to drive differentiation through the mechanical control of cell walls.

Differential spatial distribution of miR165/6 determines variability in plant root anatomy.

A clear example of interspecific variation is the number of root cortical layers in plants. The genetic mechanisms underlying this variability are poorly understood, partly because of the lack of a convenient model. Here, we demonstrate that Cardamine hirsuta, unlike Arabidopsis thaliana, has two cortical layers that are patterned during late embryogenesis. We show that a miR165/6-dependent distribution of the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) controls this pattern. Our findings reveal that interspecies variation in miRNA distribution can determine differences in anatomy in plants.

Dissecting mechanisms in root growth from the transition zone perspective.

The root of the plant Arabidopsis thaliana is a dynamic structure in which cells continuously divide and differentiate to sustain its postembryonic undetermined growth. Cells at different developmental stages are organized in distinguished zones whose position and activities are maintained constant during root growth. In this review, we will discuss the latest discoveries on the regulatory networks involved in root zonation and, in particular, in the mechanisms involved in maintaining the position of the transition zone, a root developmental boundary. Developmental boundaries physically divide cells with different functions and identities. The transition zone separates dividing cells from differentiating cells in two functional domains, preserving their identity during root growth and thus controlling root development.

miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root.

In multicellular organisms, a stringent control of the transition between cell division and differentiation is crucial for correct tissue and organ development. In the Arabidopsis root, the boundary between dividing and differentiating cells is positioned by the antagonistic interaction of the hormones auxin and cytokinin. Cytokinin affects polar auxin transport, but how this impacts the positional information required to establish this tissue boundary, is still unknown. By combining computational modeling with molecular genetics, we show that boundary formation is dependent on cytokinin's control on auxin polar transport and degradation. The regulation of both processes shapes the auxin profile in a well-defined auxin minimum. This auxin minimum positions the boundary between dividing and differentiating cells, acting as a trigger for this developmental transition, thus controlling meristem size.

Inhibition of Polycomb Repressive Complex 2 activity reduces trimethylation of H3K27 and affects development in Arabidopsis seedlings.

BACKGROUND: Polycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported. RESULTS: We show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. CONCLUSIONS: Our results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development.

A SCARECROW-based regulatory circuit controls Arabidopsis thaliana meristem size from the root endodermis.

MAIN CONCLUSION: SCARECROW controls Arabidopsis root meristem size from the root endodermis tissue by regulating the DELLA protein RGA that in turn mediates the regulation of ARR1 levels at the transition zone. Coherent organ growth requires a fine balance between cell division and cell differentiation. Intriguingly, plants continuously develop organs post-embryonically thanks to the activity of meristems that allow growth and environmental plasticity. In Arabidopsis thaliana, continued root growth is assured when division of the distal stem cell and their daughters is balanced with cell differentiation at the meristematic transition zone (TZ). We have previously shown that at the TZ, the cytokinin-dependent transcription factor ARR1 controls the rate of differentiation commitment of meristematic cells and that its activities are coordinated with those of the distal stem cells by the gene SCARECROW (SCR). In the stem cell organizer (the quiescent center, QC), SCR directly suppresses ARR1 both sustaining stem cell activities and titrating non-autonomously the ARR1 transcript levels at the TZ via auxin. Here, we show that SCR also exerts a fine control on ARR1 levels at the TZ from the endodermis by sustaining gibberellin signals. From the endodermis, SCR controls the RGA REPRESSOR OF ga1-3 (RGA) DELLA protein stability throughout the root meristem, thus controlling ARR1 transcriptional activation at the TZ. This guarantees robustness and fineness to the control of ARR1 levels necessary to balance cell division to cell differentiation in sustaining coherent root growth. Therefore, this work advances the state of the art in the field of root meristem development by integrating the activity of three hormones, auxin, gibberellin, and cytokinin, under the control of different tissue-specific activities of a single root key regulator, SCR.

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.

Proline affects the size of the root meristematic zone in Arabidopsis.

BACKGROUND: We reported previously that root elongation in Arabidopsis is promoted by exogenous proline, raising the possibility that this amino acid may modulate root growth. RESULTS: To evaluate this hypothesis we used a combination of genetic, pharmacological and molecular analyses, and showed that proline specifically affects root growth by modulating the size of the root meristem. The effects of proline on meristem size are parallel to, and independent from, hormonal pathways, and do not involve the expression of genes controlling cell differentiation at the transition zone. On the contrary, proline appears to control cell division in early stages of postembryonic root development, as shown by the expression of the G2/M-specific CYCLINB1;1 (CYCB1;1) gene. CONCLUSIONS: The overall data suggest that proline can modulate the size of root meristematic zone in Arabidopsis likely controlling cell division and, in turn, the ratio between cell division and cell differentiation.

Plant hormone cross-talk: the pivot of root growth.

Root indeterminate growth and its outstanding ability to produce new tissues continuously make this organ a highly dynamic structure able to respond promptly to external environmental stimuli. Developmental processes therefore need to be finely tuned, and hormonal cross-talk plays a pivotal role in the regulation of root growth. In contrast to what happens in animals, plant development is a post-embryonic process. A pool of stem cells, placed in a niche at the apex of the meristem, is a source of self-renewing cells that provides cells for tissue formation. During the first days post-germination, the meristem reaches its final size as a result of a balance between cell division and cell differentiation. A complex network of interactions between hormonal pathways co-ordinates such developmental inputs. In recent years, by means of molecular and computational approaches, many efforts have been made aiming to define the molecular components of these networks. In this review, we focus our attention on the molecular mechanisms at the basis of hormone cross-talk during root meristem size determination.

The CHD3 chromatin remodeler PICKLE and polycomb group proteins antagonistically regulate meristem activity in the Arabidopsis root.

The chromatin modifying Polycomb group (PcG) and trithorax group (trxG) proteins are central regulators of cell identity that maintain a tightly controlled balance between cell proliferation and cell differentiation. The opposing activities of PcG and trxG proteins ensure the correct expression of specific transcriptional programs at defined developmental stages. Here, we report that the chromatin remodeling factor PICKLE (PKL) and the PcG protein CURLY LEAF (CLF) antagonistically determine root meristem activity. Whereas loss of PKL function caused a decrease in meristematic activity, loss of CLF function increased meristematic activity. Alterations of meristematic activity in pkl and clf mutants were not connected with changes in auxin concentration but correlated with decreased or increased expression of root stem cell and meristem marker genes, respectively. Root stem cell and meristem marker genes are modified by the PcG-mediated trimethylation of histone H3 on lysine 27 (H3K27me3). Decreased expression levels of root stem cell and meristem marker genes in pkl correlated with increased levels of H3K27me3, indicating that root meristem activity is largely controlled by the antagonistic activity of PcG proteins and PKL.

Proteomics in deciphering the auxin commitment in the Arabidopsis thaliana root growth.

The development of plant root systems is characterized by a high plasticity, made possible by the continual propagation of new meristems. Root architecture is fundamental for overall plant growth, abiotic stress resistance, nutrient uptake, and response to environmental changes. Understanding the function of genes and proteins that control root architecture and stress resistance will contribute to the development of more sustainable systems of intensified crop production. To meet these challenges, proteomics provide the genome-wide scale characterization of protein expression pattern, subcellular localization, post-translational modifications, activity regulation, and molecular interactions. In this review, we describe a variety of proteomic strategies that have been applied to study the proteome of the whole organ and of specific cell types during root development. Each has advantages and limitations, but collectively they are providing important insights into the mechanisms by which auxin structures and patterns the root system and into the interplay between signaling networks, auxin transport and growth. The acquisition of proteomic, transcriptomic, and metabolomic data sets of the root apex on the cell scale has revealed the high spatial complexity of regulatory networks and fosters the use of new powerful proteomic tools for a full understanding of the control of root developmental processes and environmental responses.

Cytokinin-facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two-component circuitry.

Cytokinins propagate signals via multiple phosphorelays in a mechanism similar to bacterial two-component systems. In Arabidopsis, signal outputs are determined by the activation state of transcription factors termed type-B Arabidopsis response regulators (ARRs); however, their regulatory mechanisms are largely unknown. In this study, we demonstrate that the proteolysis of ARR2, a type-B ARR, modulates cytokinin signaling outputs. ARR2-hemagglutinin (HA) is rapidly degraded by cytokinin treatment, but other type-B ARRs, such as ARR1-HA, ARR10-HA, ARR12-HA and ARR18-HA, are not. ARR2 degradation is mediated by the 26S proteasome pathway, and requires cytokinin-induced phosphorylation of Asp80 residue in the receiver domain. Through mutational analysis of amino acid residues in the receiver domain, we found that substitution of Lys90 with Gly inhibits ARR2 degradation. ARR2(K90G) -HA in transgenic Arabidopsis conferred enhanced cytokinin sensitivity in various developmental processes, including primary root elongation, callus induction, leaf senescence and hypocotyl growth. ARR2(K90G) -HA increased the expression of type-A ARRs, primary cytokinin-responsive genes and indicators of signaling output in two-component circuits. Expression of ARR2(K90G) -HA from the native ARR2 promoter in the arr2-4 knock-out mutant also increased cytokinin sensitivity. In conclusion, ARR2 proteolysis is involved in the maintenance of the primary signaling output for normal developmental processes mediated by cytokinin in Arabidopsis.

Spatiotemporal changes in the role of cytokinin during root development.

The root is a dynamic system whose structure is regulated by a complex network of interactions between hormones. The primary root meristem is specified in the embryo. After germination, the primary root meristem grows and then reaches a final size that will be maintained during the life of the plant. Subsequently, secondary structures such as lateral roots and root nodules form via the re-specification of differentiated cells. Cytokinin plays key roles in the regulation of root development. Down-regulation of the cytokinin response is required for the specification of a new stem cell niche, during both embryo and lateral root development. In the root meristem, cytokinin signalling regulates the longitudinal zonation of the meristem by controlling cell differentiation. Moreover, cytokinin regulates radial patterning of root vasculature by promoting protophloem cell identity and by spatially inhibiting protoxylem formation. In this review, an effort is made to describe the known details of the role of cytokinin during root development, taking into account also the interactions between cytokinin and other hormones. Attention is given on the dynamicity of cytokinin signalling output during different developmental events. Indeed, there is much evidence that the effects of cytokinin change as organs grow, underlining the importance of the spatiotemporal specificity of cytokinin signalling.

Cytokinins.

The extraordinary variety that characterizes the living world in terms of forms and structures is the result of natural selection that allows an organism to be in perfect harmony with its environmental niche. Once a specific shape is acquired, many different factors act together to guarantee phenotypic robustness and developmental stability of the organism. Among these factors, hormones play a key role in the regulation and coordination of growth - they control the activity of a single cell, the progression to tissue organization, the development of specific organs, ending with the development of the entire body. In plants, hormones acquire yet another important role - plants, due to their sessile nature, along with the quest for robust development, rely on plastic development to adapt growth to a changing environment. Plant hormones play a crucial role in sensing and responding to different environmental stimuli, translating these inputs into specific developmental changes that adapt the plant body to the environment. Here, we will focus on cytokinins - a unique class of plant hormones - giving clues on their metabolism, on how they are perceived by cells and how cells change their activity in response to it. Most of the data presented have been derived by studies conducted on Arabidopsis thaliana, a plant used as a model system in plant science.

microRNA165 and 166 modulate response of the Arabidopsis root apical meristem to salt stress.

In plants, developmental plasticity allows for the modulation of organ growth in response to environmental cues. Being in contact with soil, roots are the first organ that responds to various types of soil abiotic stress such as high salt concentration. In the root, developmental plasticity relies on changes in the activity of the apical meristem, the region at the tip of the root where a set of self-renewing undifferentiated stem cells sustain growth. Here, we show that salt stress promotes differentiation of root meristem cells via reducing the dosage of the microRNAs miR165 and 166. By means of genetic, molecular and computational analysis, we show that the levels of miR165 and 166 respond to high salt concentration, and that miR165 and 166-dependent PHABULOSA (PHB) modulation is central to the response of root growth to this stress. Specifically, we show that salt-dependent reduction of miR165 and 166 causes a rapid increase in PHB expression and, hence, production of the root meristem pro-differentiation hormone cytokinin. Our data provide direct evidence for how the miRNA-dependent modulation of transcription factor dosage mediates plastic development in plants.

A PHABULOSA-Controlled Genetic Pathway Regulates Ground Tissue Patterning in the Arabidopsis Root.

In both animals and plants, development involves anatomical modifications. In the root of Arabidopsis thaliana, maturation of the ground tissue (GT)-a tissue comprising all cells between epidermal and vascular ones-is a paradigmatic example of these modifications, as it generates an additional tissue layer, the middle cortex (MC).1-4 In early post-embryonic phases, the Arabidopsis root GT is composed of one layer of endodermis and one of cortex. A second cortex layer, the MC, is generated by asymmetric cell divisions in about 80% of Arabidopsis primary roots, in a time window spanning from 7 to 14 days post-germination (dpg). The cell cycle regulator CYCLIN D6;1 (CYCD6;1) plays a central role in this process, as its accumulation in the endodermis triggers the formation of MC.5 The phytohormone gibberellin (GA) is a key regulator of the timing of MC formation, as alterations in its signaling and homeostasis result in precocious endodermal asymmetric cell divisions.3,6,7 However, little is known on how GAs are regulated during GT maturation. Here, we show that the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) is a master regulator of MC formation, controlling the accumulation of CYCD6;1 in the endodermis in a cell non-autonomous manner. We show that PHB activates the GA catabolic gene GIBBERELLIN 2 OXIDASE 2 (GA2ox2) in the vascular tissue, thus regulating the stability of the DELLA protein GIBBERELLIN INSENSITIVE (GAI)-a GA signaling repressor-in the root and, hence, CYCD6;1 expression in the endodermis.

Emerging role of cytokinin as a regulator of cellular differentiation.

Perhaps the most amazing feature of plants is their ability to grow and regenerate for years, sometimes even centuries. This fascinating characteristic is achieved thanks to the activity of stem cells, which reside in the shoot and root apical meristems. Stem cells function as a reserve of undifferentiated cells to replace organs and sustain postembryonic plant growth. To maintain meristem function, stem cells have to generate new cells at a rate similar to that of cells leaving the meristem and differentiating, thus achieving a balance between cell division and cell differentiation. Recent findings have improved our knowledge on the molecular mechanisms necessary to establish this balance and reveal a fundamental signaling role for the plant hormone cytokinin. Evidence has been provided to show that in the root meristem cytokinin acts in defined developmental domains to control cell differentiation rate, thus controlling root meristem size.