Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

Lateral Root Initiation and the Analysis of Gene Function Using Genome Editing with CRISPR in Arabidopsis.

Lateral root initiation is a post-embryonic process that requires the specification of a subset of pericycle cells adjacent to the xylem pole in the primary root into lateral root founder cells. The first visible event of lateral root initiation in Arabidopsis is the simultaneous migration of nuclei in neighbouring founder cells. Coinciding cell cycle activation is essential for founder cells in the pericycle to undergo formative divisions, resulting in the development of a lateral root primordium (LRP). The plant signalling molecule, auxin, is a major regulator of lateral root development; the understanding of the molecular mechanisms controlling lateral root initiation has progressed tremendously by the use of the Arabidopsis model and a continual improvement of molecular methodologies. Here, we provide an overview of the visible events, cell cycle regulators, and auxin signalling cascades related to the initiation of a new LRP. Furthermore, we highlight the potential of genome editing technology to analyse gene function in lateral root initiation, which provides an excellent model to answer fundamental developmental questions such as coordinated cell division, growth axis establishment as well as the specification of cell fate and cell polarity.

Stem cell ageing of the root apical meristem of Arabidopsis thaliana.

Plants form new organs from pluripotent stem cells throughout their lives and under changing environmental conditions. In the Arabidopsis root meristem, a pool of stem cells surrounding a stem cell organizer, named Quiescent Center (QC), gives rise to the specific root tissues. Among them, the columella stem cell niche that gives rise to the gravity-sensing columella cells has been used as a model system to study stem cell regulation at the young seedling stage. However, little is known about the changes of the stem cell niche during later development. Here, we report that the columella stem cell niche undergoes pronounced histological and molecular reorganization as the plant progresses towards the adult stage. Commonly-used reporters for cellular states undergo re-patterning after an initial juvenile meristem phase. Furthermore, the responsiveness to the plant hormone abscisic acid, an integrator of stress response, strongly decreases. Many ageing effects are reminiscent of the loss-of-function phenotype of the central stem cell regulator WOX5 and can be explained by gradually decreasing WOX5 expression levels during ageing. Our results show that the architecture and central regulatory components of the root stem cell niche are already highly dynamic within the first weeks of development.

Calcium spikes, waves and oscillations in plant development and biotic interactions.

The calcium ion (Ca2+) is a universal signal in all eukaryotic cells. A fundamental question is how Ca2+, a simple cation, encodes complex information with high specificity. Extensive research has established a two-step process (encoding and decoding) that governs the specificity of Ca2+ signals. While the encoding mechanism entails a complex array of channels and transporters, the decoding process features a number of Ca2+ sensors and effectors that convert Ca2+ signals into cellular effects. Along this general paradigm, some signalling components may be highly conserved, but others are divergent among different organisms. In plant cells, Ca2+ participates in numerous signalling processes, and here we focus on the latest discoveries on Ca2+-encoding mechanisms in development and biotic interactions. In particular, we use examples such as polarized cell growth of pollen tube and root hair in which tip-focused Ca2+ oscillations specify the signalling events for rapid cell elongation. In plant-microbe interactions, Ca2+ spiking and oscillations hold the key to signalling specificity: while pathogens elicit cytoplasmic spiking, symbiotic microorganisms trigger nuclear Ca2+ oscillations. Herbivore attacks or mechanical wounding can trigger Ca2+ waves traveling a long distance to transmit and convert the local signal to a systemic defence program in the whole plant. What channels and transporters work together to carve out the spatial and temporal patterns of the Ca2+ fluctuations? This question has remained enigmatic for decades until recent studies uncovered Ca2+ channels that orchestrate specific Ca2+ signatures in each of these processes. Future work will further expand the toolkit for Ca2+-encoding mechanisms and place Ca2+ signalling steps into larger signalling networks.

Root cap size and shape influence responses to the physical strength of the growth medium in Arabidopsis thaliana primary roots.

During the progression of root in soil, root cap cells are the first to encounter obstacles and are known to sense environmental cues, thus making the root cap a potential mechanosensing site. In this study, a two-layered growth medium system was developed in order to study root responses to variations in the physical strength of the medium and the importance of the root cap in the establishment of these responses. Root growth and trajectory of primary roots of Arabidopsis seedlings were investigated using in vivo image analysis. After contact with the harder layer of the medium, the root either penetrated it or underwent rapid curvature, thus enabling reorientation of growth. We initially hypothesized that the root-cap structure would affect apex penetration and reorientation, with pointed caps facilitating and domed caps impeding root penetration. This hypothesis was investigated by analysing the responses of Arabidopsis mutants with altered root caps. The primary root of lines of the fez-2 mutant, which has fewer root-cap cell layers and a more pointed root cap than wild-type roots, showed impaired penetration ability. Conversely, smb-3 roots, which display a rectangular-shaped cap, showed enhanced penetration abilities. These results, which contradict our original hypothesis, reveal a role for resistance to buckling in determining root penetration abilities.

Augmentation of root gravitropism by hypocotyl curvature in Brassica rapa seedlings.

Main Conclusion Root gravitropism of primary roots is assisted by curvature of the hypocotyl base. Root gravitropism is typically described as the sequence of signal perception, signal processing, and response that causes differential elongation and the establishment of a new gravitropic set-point angle. We describe two components of the graviresponse of Brassica seedlings that comprise a primary curvature of the root tip and a later onset but stronger curvature that occurs at the base of the hypocotyl. This second curvature is preceded by straightening of the tip region but leads to the completion of the alignment of the root axis. Curvature in both regions require a minimum displacement of 20 deg. The rate of tip curvature is a function of root length. After horizontal reorientation, tip curvature of five mm long roots curved twice as fast as 10 mm long roots (33.6 ±â€¯3.3 vs. 14.3 ±â€¯1.5 deg hr-1). The onset of curvature at the hypocotyl base is correlated with root length, but the rate of this curvature is independent of seedling length. Decapping of roots prevented tip curvature but the curvature at base of hypocotyl was unaffected. Endodermal cells at the root shoot junction show numerous, large and sedimenting amyloplasts, which likely serve as gravity sensors (statoliths). The amyloplasts at the hypocotyl were 3-4 µm in diameter, similar in size to those in the root cap, and twice the size of starch grains in the cortical layers of hypocotyl or elsewhere in the root. These data indicate that the root shoot reorientation of young seedlings is not limited to the root tip but includes more than one gravitropically responsive region.

Aluminium-induced cell death requires upregulation of NtVPE1 gene coding vacuolar processing enzyme in tobacco (Nicotiana tabacum L.).

Cell death mechanism triggered by aluminium (Al) ion was investigated at root apex of tobacco (cultivar Bright Yellow) and in cultured tobacco cell line BY-2 derived from Bright Yellow, focusing on VPE genes (NtVPE1a, NtVPE1b, NtVPE2, NtVPE3). Cell death was detected as a loss of integrity of the plasma membrane by vital staining with fluorescein diacetate (in root apex) and Evans blue (in BY-2), respectively. At root apex, the upregulation of gene expression of VPE1a and VPE1b was observed significantly after 9h of Al exposure in parallel with an enhancement of cell death, while the upregulation of VPE2 and VPE3 were observed later. Similarly, in BY-2 cells, the upregulation of VPE1a and VPE1b and the enhancement of cell death were synchronously observed after 3-h exposure to Al, while the upregulation of VPE2 and VPE3 occurred later. RNA interference (RNAi) lines of each of the VPEs were constructed in BY-2 cells. Comparative studies between wild-type and the RNAi lines indicated that both Al-enhanced VPE activity and Al-induced cell death were significantly suppressed in the RNAi lines of VPE1 (dual suppressor of VPE1a and VPE1b), but not in the RNAi lines of VPE2 and that of VPE3. Taken together, we conclude that the upregulation of VPE1 gene expression and following enhancement of VPE activity under Al stress cause cell death in actively growing or elongating cells of tobacco.

Dynamic control of lateral root positioning.

In dicot root systems, lateral roots are in general regularly spaced along the longitudinal axis of the primary root to facilitate water and nutrient uptake. Recently, recurrent programmed cell death in the root cap of the growing root has been implicated in lateral root spacing. The root cap contains an auxin source that modulates lateral root patterning. Periodic release of auxin by dying root cap cells seems to trigger lateral root specification at regular intervals. However, it is currently unclear through which molecular mechanisms auxin restricts lateral root specification to specific cells along the longitudinal and radial axes of the root, or how environmental signals impact this process.

Control of root cap maturation and cell detachment by BEARSKIN transcription factors in Arabidopsis.

The root cap supports root growth by protecting the root meristem, sensing gravity and interacting with the rhizosphere through metabolite secretion and cell dispersal. Sustained root cap functions therefore rely on balanced proliferation of proximal stem cells and regulated detachment of distal mature cells. Although the gene regulatory network that governs stem cell activity in the root cap has been extensively studied in Arabidopsis, the mechanisms by which root cap cells mature and detach from the root tip are poorly understood. We performed a detailed expression analysis of three regulators of root cap differentiation, SOMBRERO, BEARSKIN1 and BEARSKIN2, and identified their downstream genes. Our results indicate that expression of BEARSKIN1 and BEARSKIN2 is associated with cell positioning on the root surface. We identified a glycosyl hydrolase 28 (GH28) family polygalacturonase (PG) gene as a direct target of BEARSKIN1. Overexpression and loss-of-function analyses demonstrated that the protein encoded by this PG gene facilitates cell detachment. We thus revealed a molecular link between the key regulators of root cap differentiation and the cellular events underlying root cap-specific functions.

The build-up of osmotic stress responses within the growing root apex using kinematics and RNA-sequencing.

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth.

Cyclic programmed cell death stimulates hormone signaling and root development in Arabidopsis.

The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.

The root cap: a short story of life and death.

Over 130 years ago, Charles Darwin recognized that sensory functions in the root tip influence directional root growth. Modern plant biology has unravelled that many of the functions that Darwin attributed to the root tip are actually accomplished by a particular organ-the root cap. The root cap surrounds and protects the meristematic stem cells at the growing root tip. Due to this vanguard position, the root cap is predisposed to receive and transmit environmental information to the root proper. In contrast to other plant organs, the root cap shows a rapid turnover of short-lived cells regulated by an intricate balance of cell generation, differentiation, and degeneration. Thanks to these particular features, the root cap is an excellent developmental model system, in which generation, differentiation, and degeneration of cells can be investigated in a conveniently compact spatial and temporal frame. In this review, we give an overview of the current knowledge and concepts of root cap biology, focusing on the model plant Arabidopsis thaliana.

PCaP2 regulates nuclear positioning in growing Arabidopsis thaliana root hairs by modulating filamentous actin organization.

KEY MESSAGE: PCaP2 plays a key role in maintaining the nucleus at a relatively fixed distance from the apex during root hair growth by modulating actin filaments. During root hair growth, the nucleus localizes at a relatively fixed distance from the apex. In Arabidopsis thaliana, the position of the nucleus is mainly dependent on the configuration of microfilaments (filamentous actin). However, the mechanisms underlying the regulation of actin dynamics and organization for nuclear positioning are largely unknown. In the present study, we demonstrated that plasma membrane-associated Ca(2+) binding protein 2 (PCaP2) influences the position of the nucleus during root hair growth. Abnormal expression of PCaP2 in pcap2 and PCaP2 over-expression plants led to the disorganization of actin filaments, rather than microtubules, in the apex and sub-apical regions of root hairs, which resulted in aberrant root hair growth patterns and misplaced nuclei. Analyses using a PCaP2 mutant protein revealed that actin-severing activity is essential for the function of PCaP2 in root hairs. We demonstrated that PCaP2 plays a key role in maintaining nuclear position in growing root hairs by modulating actin filaments.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Programmed cell death: new role in trimming the root tips.

How is a rapid cellular turnover of the lateral root cap achieved in plants to control cap size in the growing root tips? Downstream of ANAC033/SOMBRERO, a highly organized and temporally coordinated cell death program involving BFN1 nuclease-mediated rapid corpse clearance eliminates these cells.

Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.

BACKGROUND: The root cap is a plant organ that ensheathes the meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing constant cell generation by specific stem cells with the disposal of differentiated cells at the root cap edge. This cellular turnover is critical for the maintenance of root cap size and its position around the growing root tip, but how this is achieved and controlled in the model plant Arabidopsis thaliana remains subject to contradictory hypotheses. RESULTS: Here, we show that a highly organized cell death program is the final step of lateral root cap differentiation and that preparation for cell death is transcriptionally controlled by ANAC033/SOMBRERO. Precise timing of cell death is critical for the elimination of root cap cells before they fully enter the root elongation zone, which in turn is important in order to allow optimal root growth. Root cap cell death is followed by a rapid cell-autonomous corpse clearance and DNA fragmentation dependent on the S1-P1 type nuclease BFN1. CONCLUSIONS: Based on these results, we propose a novel concept in plant development that recognizes programmed cell death as a mechanism for maintaining organ size and tissue homeostasis in the Arabidopsis root cap.

The receptor-like kinases GSO1 and GSO2 together regulate root growth in Arabidopsis through control of cell division and cell fate specification.

BACKGROUND: The root apical meristem of Arabidopsis is established post-embryonically as the main source of root cells, and its activity is maintained by complex bidirectional signaling between stem cells and mature cells. The receptor-like kinases GASSHO1 (GSO1) and GSO2 have been shown to regulate aerial epidermal function and seedling growth in Arabidopsis. RESULTS: Here we show that gso1; gso2 seedlings also have root growth and patterning defects. Analyses of mutant root morphology indicate abnormal numbers of cells in longitudinal files and radial cell layers, as well as aberrant stem cell division planes. gso1; gso2 double mutants misexpress markers for stem cells and differentiated root cell types. In addition, gso1; gso2 root growth defects, but not marker missexpression or patterning phenotypes, are rescued by growth on media containing metabolizable sugars. CONCLUSIONS: We conclude that GSO1 and GSO2 function together in intercellular signaling to positively regulate cell proliferation, differentiation of root cell types, and stem cell identity. In addition, GSO1 and GSO2 control seedling root growth by modulating sucrose response after germination.

Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3.

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem.

Sucrose transport is inhibited by okadaic acid during regeneration of sugar-starved Vicia faba root meristem cells.

The sucrose-induced resumption of cell cycle in the Vicia faba root meristem cells, blocked in two principal control points PCP1/2 by carbohydrate starvation, occurs after 12 h of metabolic regeneration comprising increased activity of sucrose synthase (SuSy) and hexokinase (HK) as well as starch grain and cell wall matrix polysaccharide biosynthesis. Okadaic acid (OA), the specific protein phosphatase 1/2A inhibitor, supplied at the beginning of the recovery period (0-3 h) completely blocks these processes, making cell cycle resumption impossible. On the other hand, when added at the end (9-12 h), OA has a weak inhibitory effect. The aim of these studies was: (1) to establish how sucrose is transported into the cells and whether the above-mentioned effects are correlated with the intensity of its uptake at the beginning and at the end of the metabolic regeneration; and (2) to determine whether OA, blocking sucrose metabolism, also interferes with the process of sucrose uptake and distribution. The level of [(3)H]sucrose uptake was measured by liquid scintillation counting while sugar distribution was analyzed using microautoradiography and electron microscopy. The results showed that sucrose entered the meristematic cells along symplastic or apoplastic pathways and, to a lesser extent, through endocytosis. The cytoplasmic compartments (endoplasmic reticulum, vacuoles, plastids) and the nucleus were labeled. The intensity of [(3)H]sucrose uptake was nearly 2-fold lower during the initial than during the final period of metabolic regeneration. OA inhibited the apoplastic pathway of radioactive molecule uptake and its distribution between cell compartments, implicating PP1/2A involvement in the regulation of this transport.

A method to determine the displacement velocity field in the apical region of the Arabidopsis root.

In angiosperms, growth of the root apex is determined by the quiescent centre. All tissues of the root proper and the root cap are derived from initial cells that surround this zone. The diversity of cell lineages originated from these initials suggests an interesting variation of the displacement velocity within the root apex. However, little is known about this variation, especially in the most apical region including the root cap. This paper shows a method of determination of velocity field for this region taking the Arabidopsis root apex as example. Assuming the symplastic growth without a rotation around the root axis, the method combines mathematical modelling and two types of empirical data: the published velocity profile along the root axis above the quiescent centre, and dimensions of cell packet originated from the initials of epidermis and lateral root cap. The velocities, calculated for points of the axial section, vary in length and direction. Their length increases with distance from the quiescent centre, in the root cap at least twice slower than in the root proper, if points at similar distance from the quiescent centre are compared. The vector orientation depends on the position of a calculation point, the widest range of angular changes, reaching almost 90°, in the lateral root cap. It is demonstrated how the velocity field is related to both distribution of growth rates and growth-resulted deformation of the cell wall system. Also changes in the field due to cell pattern asymmetry and differences in slope of the velocity profile are modelled.