Plant Growth Regulation in Cell and Tissue Culture In Vitro.

Precise knowledge of all aspects controlling plant tissue culture and in vitro plant regeneration is crucial for plant biotechnologists and their correlated industry, as there is increasing demand for this scientific knowledge, resulting in more productive and resilient plants in the field. However, the development and application of cell and tissue culture techniques are usually based on empirical studies, although some data-driven models are available. Overall, the success of plant tissue culture is dependent on several factors such as available nutrients, endogenous auxin synthesis, organic compounds, and environment conditions. In this review, the most important aspects are described one by one, with some practical recommendations based on basic research in plant physiology and sharing our practical experience from over 20 years of research in this field. The main aim is to help new plant biotechnologists and increase the impact of the plant tissue culture industry worldwide.

Regulation of early seedling establishment and root development in Arabidopsis thaliana by light and carbohydrates.

MAIN CONCLUSION: Root development is regulated by sucrose and light during early seedling establishment through changes in the auxin response and chromatin topology. Light is a key environmental signal that regulates plant growth and development. The impact of light on development is primarily analyzed in the above-ground tissues, but little is known about the mechanisms by which light shapes the architecture of underground roots. Our study shows that carbohydrate starvation during skotomorphogenesis is accompanied by compaction of nuclei in the root apical meristem, which prevents cell cycle progression and leads to irreversible root differentiation in the absence of external carbohydrates, as evidenced by the lack of DNA replication and increased numbers of nuclei with specific chromatin characteristics. In these conditions, induction of photomorphogenesis was unable to restore seedling growth, as overall root growth was compromised. The addition of carbohydrates, either locally or systemically by transferring seedlings to sugar-containing medium, led to the induction of adventitious root formation with rapid recovery of seedling growth. Conversely, transferring in vitro carbohydrate-grown seedlings from light to dark transiently promoted cell elongation and significantly reduced root meristem size, but did not primarily affect cell cycle kinetics. We show that, in the presence of sucrose, dark incubation does not affect zonation in the root apical meristem but leads to shortening of the proliferative and transition zones. Sugar starvation led to a rapid increase in lysine demethylation of histone H3 at position K9, which preceded a rapid decline in cell cycle activity and activation of cell differentiation. In conclusion, carbohydrates are required for cell cycle activity, epigenetics reprogramming and for postmitotic cell elongation and auxin-regulated response in the root apical meristem.

Optimization of Callus Induction and Shoot Regeneration from Tomato Cotyledon Explants.

Cultivated tomato (Solanum lycopersicum L.) is one of the most important horticultural crops in the world. The optimization of culture media for callus formation and tissue regeneration of different tomato genotypes presents numerous biotechnological applications. In this work, we have analyzed the effect of different concentrations of zeatin and indole-3-acetic acid on the regeneration of cotyledon explants in tomato cultivars M82 and Micro-Tom. We evaluated regeneration parameters such as the percentage of callus formation and the area of callus formed, as well as the initiation percentage and the number of adventitious shoots. The best hormone combination produced shoot-like structures after 2-3 weeks. We observed the formation of leaf primordia from these structures after about 3-4 weeks. Upon transferring the regenerating micro-stems to a defined growth medium, it was possible to obtain whole plantlets between 4 and 6 weeks. This hormone combination was applied to other genotypes of S. lycopersicum, including commercial varieties and ancestral tomato varieties. Our method is suitable for obtaining many plantlets of different tomato genotypes from cotyledon explants in a very short time, with direct applications for plant transformation, use of gene editing techniques, and vegetative propagation of elite cultivars.

AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response.

An environmentally responsive root system is crucial for plant growth and crop yield, especially in suboptimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density while simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture. Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here, we describe a search for proteins that regulate root system architecture (RSA) by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co-purifying proteins. Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here, we show that, in Arabidopsis, AZG1 is a cytokinin (CK) import protein that co-localizes with and stabilizes PIN1, linking auxin and CK transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1-dependent under salt stress. This report therefore identifies a potential point for auxin:cytokinin crosstalk, which shapes RSA in response to NaCl.

Role of reactive oxygen species in the modulation of auxin flux and root development in Arabidopsis thaliana.

Reactive oxygen species (ROS) play a dual role in plant biology, acting as important signal transduction molecules and as toxic byproducts of aerobic metabolism that accumulate in cells upon exposure to different stressors and lead to cell death. In plants, root architecture is regulated by the distribution and intercellular flow of the phytohormone auxin. In this study, we identified ROS as an important modulator of auxin distribution and response in the root. ROS production is necessary for root growth, proper tissue patterning, cell growth, and lateral root (LR) induction. Alterations in ROS balance led to altered auxin distribution and response in SOD and RHD2 loss-of-function mutants. Treatment of Arabidopsis seedlings with additional sources of ROS (hydrogen peroxide) or an ROS production inhibitor (diphenylene iodonium) induced phenocopies of the mutants studied. Simultaneous application of auxin and ROS increased LR primordia induction, and PIN-FORMED protein immunolocalization further demonstrated the existing link between auxin and ROS in orchestrating cell division and auxin flux during root development. In Arabidopsis roots, genetic alterations in ROS balance led to defective auxin distribution and growth-related responses in roots. Exogenous hydrogen peroxide alters the establishment of the endogenous auxin gradient in the root meristem through regulation of PIN-FORMED polarity, while the simultaneous application of hydrogen peroxide and auxin enhanced LR induction in a dose- and position-dependent manner through activation of cell division.

Mapping of the Classical Mutation rosette Highlights a Role for Calcium in Wound-Induced Rooting.

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming.

A simple pipeline for cell cycle kinetic studies in the root apical meristem.

Root system architecture ultimately depends on precise signaling between different cells and tissues in the root apical meristem (RAM) and integration with environmental cues. This study describes a simple pipeline to simultaneously determine cellular parameters, nucleus geometry, and cell cycle kinetics in the RAM. The method uses marker-free techniques for nucleus and cell boundary detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining for DNA replication quantification. Based on this approach, we characterized differences in cell volume, nucleus volume, and nucleus shape across different domains of the Arabidopsis RAM. We found that DNA replication patterns were cell layer and region dependent. G2 phase duration, which varied from 3.5 h in the pericycle to more than 4.5 h in the epidermis, was found to be associated with some features of nucleus geometry. Endocycle duration was determined as the time required to achieve 100% EdU-positive cells in the elongation zone and, as such, it was estimated to be in the region of 5 h for the epidermis and cortex. This experimental pipeline could be used to precisely map cell cycle duration in the RAM of mutants and in response to environmental stress in several plant species without the need for introgressing molecular cell cycle markers.

The role of AUX1 during lateral root development in the domestication of the model C4 grass Setaria italica.

C4 photosynthesis increases the efficiency of carbon fixation by spatially separating high concentrations of molecular oxygen from Rubisco. The specialized leaf anatomy required for this separation evolved independently many times. The morphology of C4 root systems is also distinctive and adapted to support high rates of photosynthesis; however, little is known about the molecular mechanisms that have driven the evolution of C4 root system architecture. Using a mutant screen in the C4 model plant Setaria italica, we identify Siaux1-1 and Siaux1-2 as root system architecture mutants. Unlike in S. viridis, AUX1 promotes lateral root development in S. italica. A cell by cell analysis of the Siaux1-1 root apical meristem revealed changes in the distribution of cell volumes in all cell layers and a dependence of the frequency of protophloem and protoxylem strands on SiAUX1. We explore the molecular basis of the role of SiAUX1 in seedling development using an RNAseq analysis of wild-type and Siaux1-1 plants and present novel targets for SiAUX1-dependent gene regulation. Using a selection sweep and haplotype analysis of SiAUX1, we show that Hap-2412TT in the promoter region of SiAUX1 is an allele which is associated with lateral root number and has been strongly selected for during Setaria domestication.

Methods of In Situ Quantitative Root Biology.

When dealing with plant roots, a multiscale description of the functional root structure is needed. Since the beginning of 21st century, new devices such as laser confocal microscopes have been accessible for coarse root structure measurements, including three-dimensional (3D) reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitative determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for 3D analysis of root features at single-cell resolution, including root asymmetry, lateral root analysis, cell size and nuclear organization, cell-cycle kinetics, and chromatin structure analysis. Quantitative maps of the root apical meristem (RAM) are shown for different species, including Arabidopsis thaliana (L.), Heynh, Nicotiana tabacum L., Medicago sativa L., and Setaria italica (L.) P. Beauv. The 3D analysis of the RAM in these species showed divergence in chromatin organization and cell volume distribution that might be used to study root zonation for each root tissue. Detailed protocols and possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference.

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation.

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Cell Dynamics in WOX5-Overexpressing Root Tips: The Impact of Local Auxin Biosynthesis.

Root stem cell niche functioning requires the formation and maintenance of the specific "auxin-rich domain" governed by directional auxin transport and local auxin production. Auxin maximum co-localizes with the WOX5 expression domain in the quiescent center that separates mitotically active proximal and distal root meristems. Here we unravel the interconnected processes happening under WOX5 overexpression by combining in vivo experiments and mathematical modeling. We showed that WOX5-induced TAA1-mediated auxin biosynthesis is the cause, whereas auxin accumulation, PIN transporters relocation, and auxin redistribution between proximal and distal root meristems are its subsequent effects that influence the formation of the well-described phenotype with an enlarged root cap. These findings helped us to clarify the role of WOX5, which serves as a local QC-specific regulator that activates biosynthesis of non-cell-autonomous signal auxin to regulate the distal meristem functioning. The mathematical model with WOX5-mediated auxin biosynthesis and auxin-regulated cell growth, division, and detachment reproduces the columella cells dynamics in both wild type and under WOX5 dysregulation.

Glutathione Enhances Auxin Sensitivity in Arabidopsis Roots.

Root development is regulated by the tripeptide glutathione (GSH), a strong non-enzymatic antioxidant found in plants but with a poorly understood function in roots. Here, Arabidopsis mutants deficient in GSH biosynthesis (cad2, rax1, and rml1) and plants treated with the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) showed root growth inhibition, significant alterations in the root apical meristem (RAM) structure (length and cell division), and defects in lateral root formation. Investigation of the molecular mechanisms of GSH action showed that GSH deficiency modulated total ubiquitination of proteins and inhibited the auxin-related, ubiquitination-dependent degradation of Aux/IAA proteins and the transcriptional activation of early auxin-responsive genes. However, the DR5 auxin transcriptional response differed in root apical meristem (RAM) and pericycle cells. The RAM DR5 signal was increased due to the up-regulation of the auxin biosynthesis TAA1 protein and down-regulation of PIN4 and PIN2, which can act as auxin sinks in the root tip. The transcription auxin response (the DR5 signal and expression of auxin responsive genes) in isolated roots, induced by a low (0.1 µM) auxin concentration, was blocked following GSH depletion of the roots by BSO treatment. A higher auxin concentration (0.5 µM) offset this GSH deficiency effect on DR5 expression, indicating that GSH deficiency does not completely block the transcriptional auxin response, but decreases its sensitivity. The ROS regulation of GSH, the active GSH role in cell proliferation, and GSH cross-talk with auxin assume a potential role for GSH in the modulation of root architecture under stress conditions.

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming.

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.

3D Analysis of Mitosis Distribution Pattern in the Plant Root Tip with iRoCS Toolbox.

The protocol allows to define and characterize mitosis distribution patterns in the plant root meristem. The method does not require genetic markers, which makes it applicable to plants of different non-transgenic genotypes, including ecotypes, mutants, and non-model plant species. Computer analysis of the mitosis distribution in three dimensions with iRoCS Toolbox identifies statistically significant changes in proliferation activity within specific root tissues and cell lineages.

Salicylic Acid Affects Root Meristem Patterning via Auxin Distribution in a Concentration-Dependent Manner.

The phytohormone salicylic acid (SA) is well known for its induction of pathogenesis-related proteins and systemic acquired resistance; SA also has specific effects on plant growth and development. Here we analyzed the effect of SA on Arabidopsis (Arabidopsis thaliana) root development. We show that exogenous SA treatment at low (below 50 µM) and high (greater than 50 µM) concentrations affect root meristem development in two different PR1-independent ways. Low-concentration SA promoted adventitious roots and altered architecture of the root apical meristem, whereas high-concentration SA inhibited all growth processes in the root. All exposures to exogenous SA led to changes in auxin synthesis and transport. A wide range of SA treatment concentrations activated auxin synthesis, but the effect of SA on auxin transport was dose dependent. Mathematical modeling of auxin synthesis and transport predicted auxin accumulation or depletion in the root tip following low- or high-concentration SA treatments, respectively. SA-induced auxin accumulation led to the formation of more layers of columella initials, an additional cortical cell layer (middle cortex), and extra files of epidermis, cortex, and endodermis cells. Suppression of SHORT ROOT and activation of CYCLIN D6;1 mediated the changes in radial architecture of the root. We propose that low-concentration SA plays an important role in shaping root meristem structure and root system architecture.

Interplay of the two ancient metabolites auxin and MEcPP regulates adaptive growth.

The ancient morphoregulatory hormone auxin dynamically realigns dedicated cellular processes that shape plant growth under prevailing environmental conditions. However, the nature of the stress-responsive signal altering auxin homeostasis remains elusive. Here we establish that the evolutionarily conserved plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) controls adaptive growth by dual transcriptional and post-translational regulatory inputs that modulate auxin levels and distribution patterns in response to stress. We demonstrate that in vivo accumulation or exogenous application of MEcPP alters the expression of two auxin reporters, DR5:GFP and DII-VENUS, and reduces the abundance of the auxin-efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane. However, pharmacological intervention with clathrin-mediated endocytosis blocks the PIN1 reduction. This study provides insight into the interplay between these two indispensable signaling metabolites by establishing the mode of MEcPP action in altering auxin homeostasis, and as such, positioning plastidial function as the primary driver of adaptive growth.

A PLA-iRoCS Pipeline for the Localization of Protein-Protein Interactions In Situ.

In plants as well as other organisms, protein localization alone is insufficient to provide a mechanistic link between stimulus and process regulation. This is because protein-protein interactions are central to the regulation of biological processes. However, they remain very difficult to detect in situ, with the choice of tools for the detection of protein-protein interaction in situ still in need of expansion. Here, we provide a protocol for the detection and accurate localization of protein interactions based on the combination of a whole-mount proximity ligation assay and iRoCS, a coordinate system able to standardize subtle differences between the architecture of individual Arabidopsis roots.