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.     

A matter of size: developmental control of organ size in plants

The intrinsic size of plant organs is determined by developmental signals, yet the molecular and genetic mechanisms that control organ size are largely unknown. Ongoing functional analysis of Arabidopsis genes is defining important regulators involved in these mechanisms. Key features of this control are the coordinated activation of growth and cell division by growth regulators and the maintenance of meristematic competence by the ANT gene, which acts as an organ-size checkpoint. Alterations of genome size by polyploidization and endoreduplication can reset this checkpoint by ploidy-dependent, epigenetically regulated differential gene expression. In addition, the regulation of polarized growth and phytohormone signaling also affect final organ size. These findings reveal unique aspects of plant organ-size control that are distinct from animal organ-size control.     

Size Control in Plants—Lessons from Leaves and Flowers

To achieve optimal functionality, plant organs like leaves and petals have to grow to a certain size. Beginning with a limited number of undifferentiated cells, the final size of an organ is attained by a complex interplay of cell proliferation and subsequent cell expansion. Regulatory mechanisms that integrate intrinsic growth signals and environmental cues are required to enable optimal leaf and flower development. This review focuses on plant-specific principles of growth reaching from the cellular to the organ level. The currently known genetic pathways underlying these principles are summarized and network connections are highlighted. Putative non–cell autonomously acting mechanisms that might coordinate plant-cell growth are discussed.Over millions of years, plant leaves and flowers evolved into an enormous range of shapes and sizes. Likely reflecting adaptations to changing environmental conditions, even closely related species often differ dramatically in their organ sizes (Mizukami 2001). Although interspecies diversity is remarkably high, species-specific leaf and petal characteristics are often highly uniform between individuals grown under constant conditions. This suggests that tight genetic control is used to integrate intrinsic growth signals and environmental cues to enable organ growth to a defined size. This review summarizes the current knowledge of the regulatory networks of plant size control at the cellular and at the organ level. We will focus on the regulation of determinate growth of lateral plant organs, such as simple leaves and petals.     

BIN4, a novel component of the plant DNA topoisomerase VI complex, is required for endoreduplication in Arabidopsis

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.     

Synergistic action of auxin and cytokinin mediates aluminum‐induced root growth inhibition in Arabidopsis

Auxin acts synergistically with cytokinin to control the shoot stem‐cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress‐induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al‐induced auxin signaling. Al stress triggers a local cytokinin response in the root‐apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up‐regulated specifically in the root‐apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum‐induced root growth inhibition in Arabidopsis.     

miR390, Arabidopsis TAS3 tasiRNAs,and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating Lateral Root Growth

Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.     

Proteomic Analysis Reveals That Developing Leaves are More Sensitive to Nitrogen Fertilizer Than Mature Leaves

The size of rice leaves is tightly controlled by environmental and genetic factors. Several functional genes control leaf growth and development by regulating cell expansion and cell cycle activity. The regulation of leaf growth, particularly the effects of environmental conditions on leaf size, is still poorly understood. We examined the environmental control of leaf size in rice (Oryza sativa) by performing a comparative proteomic analysis, which showed that exposure to high-nitrogen levels produced enlarged leaves. The enhanced leaf growth occurred mainly as a result of an increased number of cell cycles. Two proteins related to cell division, FtsZ and ERBB3 binding protein, were increased by nitrogen treatment in the developing leaves. The expression of a type-A response regulator, OsRR2, was also elevated in developing leaves. OsRR2 acts as a negative regulator of cytokinin signaling and may reduce the cytokinin content in developing leaves; a low cytokinin level is necessary for leaf development. By analyzing the proteome response to nitrogen in both developing and mature leaves, we provide deeper insight into the mechanism by which nitrogen treatment affects the phenotype.     

New clues to organ size control in plants

Plant growth has unparalleled importance for human civilization, yet we are only starting to gain an understanding of its mechanisms. The growth rate and final size of plant organs is determined by both genetic constraints and environmental factors. Regulatory inputs act at two control points: on proliferation; and on the transition between proliferation and differentiation. Cell-autonomous and short-range growth signals act within meristematic domains, whereas diffusible signals from differentiated parts to proliferating cells provide measures of geometry and size and channel environmental inputs.     

The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth

Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.     

Dissecting the effects of nitrate, sucrose and osmotic potential on Arabidopsis root and shoot system growth in laboratory assays

Studying the specific effects of water and nutrients on plant development is difficult because changes in a single component can often trigger multiple response pathways. Such confounding issues are prevalent in commonly used laboratory assays. For example, increasing the nitrate concentration in growth media alters both nitrate availability and osmotic potential. In addition, it was recently shown that a change in the osmotic potential of media alters the plant's ability to take up other nutrients such as sucrose. It can also be difficult to identify the initial target tissue of a particular environmental cue because there are correlated changes in development of many organs. These growth changes may be coordinately regulated, or changes in development of one organ may trigger changes in development of another organ as a secondary effect. All these complexities make analyses of plant responses to environmental factors difficult to interpret. Here, we review the literature on the effects of nitrate, sucrose and water availability on root system growth and discuss the mechanisms underlying these effects. We then present experiments that examine the impact of nitrate, sucrose and water on root and shoot system growth in culture using an approach that holds all variables constant except the one under analysis. We found that while all three factors also alter root system size, changes in sucrose and osmotic potential also altered shoot system size. In contrast, we found that, when osmotic effects are controlled, nitrate specifically inhibits root system growth while having no effect on shoot system growth. This effectively decreases the root : shoot ratio. Alterations in root : shoot ratio have been widely observed in response to nitrogen starvation, where root growth is selectively increased, but the present results suggest that alterations in this ratio can be triggered across a wide spectrum of nitrate concentrations.     

Cell-type action specificity of auxin on Arabidopsis root growth

The plant hormone auxin plays a critical role in root growth and development; however, the contributions or specific roles of cell-type auxin signals in root growth and development are not well understood. Here, we mapped tissue and cell types that are important for auxin-mediated root growth and development by manipulating the local response and synthesis of auxin. Repressing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele strongly inhibited root growth, with the largest effect observed in the endodermis. Enhancing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele also caused reduced root growth, albeit to a lesser extent. Moreover, we established that root growth was inhibited by enhancement of auxin synthesis in specific cell types of the epidermis, cortex and endodermis, whereas increased auxin synthesis in the pericycle and stele had only minor effects on root growth. Our study thus establishes an association between cellular identity and cell type-specific auxin signaling that guides root growth and development.     

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.     

RICE SALT SENSITIVE3 binding to bHLH and JAZ factors mediates control of cell wall plasticity in the root apex

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of plants, in accordance with their sessile lifestyle. This is linked to the balance between plasticity and rigidity of cells in the root apex, and thus is coordinated with the control of cell wall properties. However, mechanisms underlying such harmonization are not well understood, in particular under stressful conditions. We have recently demonstrated that RICE SALT SENSITIVE3 (RSS3), a nuclear factor that mediates restrictive expression of jasmonate-induced genes, plays an important role in root elongation under saline conditions. In this study, we report that loss-of-function of RSS3 results in changes in cell wall properties such as lignin deposition and sensitivity to a cellulose synthase inhibitor, concomitant with altered expression of genes involved in cell wall metabolism. Based on these and previous phenotypic observations of the rss3 mutant, we propose that RSS3 plays a role in the coordinated control of root elongation and cell wall plasticity in the root apex.     

Root responses to soil physical conditions; growth dynamics from field to cell

Root growth in the field is often slowed by a combination of soil physical stresses, including mechanical impedance, water stress, and oxygen deficiency. The stresses operating may vary continually, depending on the location of the root in the soil profile, the prevailing soil water conditions, and the degree to which the soil has been compacted. The dynamics of root growth responses are considered in this paper, together with the cellular responses that underlie them. Certain root responses facilitate elongation in hard soil, for example, increased sloughing of border cells and exudation from the root cap decreases friction; and thickening of the root relieves stress in front of the root apex and decreases buckling. Whole root systems may also grow preferentially in loose versus dense soil, but this response depends on genotype and the spatial arrangement of loose and compact soil with respect to the main root axes. Decreased root elongation is often accompanied by a decrease in both cell flux and axial cell extension, and recent computer-based models are increasing our understanding of these processes. In the case of mechanical impedance, large changes in cell shape occur, giving rise to shorter fatter cells. There is still uncertainty about many aspects of this response, including the changes in cell walls that control axial versus radial extension, and the degree to which the epidermis, cortex, and stele control root elongation. Optical flow techniques enable tracking of root surfaces with time to yield estimates of two-dimensional velocity fields. It is demonstrated that these techniques can be applied successfully to time-lapse sequences of confocal microscope images of living roots, in order to determine velocity fields and strain rates of groups of cells. In combination with new molecular approaches this provides a promising way of investigating and modelling the mechanisms controlling growth perturbations in response to environmental stresses.     

KLUH/CYP78A5 promotes organ growth without affecting the size of the early primordium

Mobile signals play a key role in controlling the growth of organisms. In Arabidopsis, the cytochrome P450 CYP78A5/KLUH (KLU) non-cell autonomously stimulates cell proliferation in developing organs. In a recent study, we determined the range of KLU action, using a widely applicable system to predictably generate chimaeric plants. We showed that KLU acts not only within individual floral organs or flowers, but that its overall activity level is integrated across an inflorescence to determine organ size. Here, we address the question at which stage of petal development KLU acts to promote growth. We demonstrate that the size of the very young petal primordium in klu mutants is not altered, supporting the conclusion that KLU acts during later stages of organ outgrowth and a correspondingly longer range of the presumed KLU-dependent growth signal.Key words: Arabidopsis, KLUH, floral organ growth, signaling, flower, growth coordination     

Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species

Plant growth is adaptively modulated in response to environmental change. The phytohormone gibberellin (GA) promotes growth by stimulating destruction of the nuclear growth-repressing DELLA proteins [1-7], thus providing a mechanism for environmentally responsive growth regulation [8, 9]. Furthermore, DELLAs promote survival of adverse environments [8]. However, the relationship between these survival and growth-regulatory mechanisms was previously unknown. Here, we show that both mechanisms are dependent upon control of the accumulation of reactive oxygen species (ROS). ROS are small molecules generated during development and in response to stress that play diverse roles as eukaryotic intracellular second messengers [10]. We show that Arabidopsis DELLAs cause ROS levels to remain low after either biotic or abiotic stress, thus delaying cell death and promoting tolerance. In essence, stress-induced DELLA accumulation elevates the expression of genes encoding ROS-detoxification enzymes, thus reducing ROS levels. In accord with recent demonstrations that ROS control root cell expansion [11, 12], we also show that DELLAs regulate root-hair growth via a ROS-dependent mechanism. We therefore propose that environmental variability regulates DELLA activity [8] and that DELLAs in turn couple the downstream regulation of plant growth and stress tolerance through modulation of ROS levels.