scholarly journals Live tracking of moving samples in confocal microscopy for vertically grown roots

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Daniel von Wangenheim ◽  
Robert Hauschild ◽  
Matyáš Fendrych ◽  
Vanessa Barone ◽  
Eva Benková ◽  
...  
Keyword(s):  
Moving Objects ◽  
Root Tip ◽  
Data Sets ◽  
Root Tips ◽  
Arabidopsis Root ◽  
High Resolution Data ◽  
Environmental Signals ◽  
Biological Studies ◽  
Custom Software ◽  
Sample Position

Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker – a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high-resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes.

scholarly journals Live tracking of Moving samples in confocal microscopy for vertically grown roots tips

2017 ◽  
Author(s):  
Daniel von Wangenheim ◽  
Robert Hauschild ◽  
Matyáš Fendrych ◽  
Vanessa Barone ◽  
Jiří Friml
Keyword(s):  
Zebrafish Embryo ◽  
Moving Objects ◽  
Root Tip ◽  
Data Sets ◽  
Root Tips ◽  
High Resolution Data ◽  
Environmental Signals ◽  
Biological Studies ◽  
Custom Software ◽  
Sample Position

AbstractRoots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker - a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes.

Nitrate and glutamate sensing by plant roots

2005 ◽  
Vol 33 (1) ◽  
pp. 283-286 ◽  
Author(s):  
S. Filleur ◽  
P. Walch-Liu ◽  
Y. Gan ◽  
B.G. Forde
Keyword(s):  
Root Growth ◽  
Lateral Root ◽  
Lateral Roots ◽  
Primary Root ◽  
Root Tip ◽  
Root Tips ◽  
Environmental Signals ◽  
Specific Effects ◽  
Large Numbers ◽  
Low Concentrations

The architecture of a root system plays a major role in determining how efficiently a plant can capture water and nutrients from the soil. Growth occurs at the root tips and the process of exploring the soil volume depends on the behaviour of large numbers of individual root tips at different orders of branching. Each root tip is equipped with a battery of sensory mechanisms that enable it to respond to a range of environmental signals, including nutrients, water potential, light, gravity and touch. We have previously identified a MADS (MCM1, agamous, deficiens and SRF) box gene (ANR1) in Arabidopsis thaliana that is involved in modulating the rate of lateral root growth in response to changes in the external NO3− supply. Transgenic plants have been generated in which a constitutively expressed ANR1 protein can be post-translationally activated by treatment with dexamethasone (DEX). When roots of these lines are treated with DEX, lateral root growth is markedly stimulated but there is no effect on primary root growth, suggesting that one or more components of the regulatory pathway that operate in conjunction with ANR1 in lateral roots may be absent in the primary root tip. We have recently observed some very specific effects of low concentrations of glutamate on root growth, resulting in significant changes in root architecture. Experimental evidence suggests that this response involves the sensing of extracellular glutamate by root tip cells. We are currently investigating the possible role of plant ionotropic glutamate receptors in this sensory mechanism.

scholarly journals Metaphloem development in the Arabidopsis root tip

2021 ◽  
Author(s):  
Moritz Graeff ◽  
Christian Hardtke
Keyword(s):  
Terrestrial Ecosystems ◽  
Phloem Transport ◽  
Developmental Trajectory ◽  
Root Tip ◽  
Root Tips ◽  
Arabidopsis Root ◽  
Sieve Elements ◽  
Evolutionary Innovation ◽  
Spatio Temporal ◽  
Cell Niche

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early, so-called protophloem. This is easily observed in Arabidopsis root meristems, where the differentiation of individual protophloem sieve element precursors into interconnected, conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared to protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here we describe an improved protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of new molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

scholarly journals Metaphloem development in the Arabidopsis root tip

Development ◽  
2021 ◽  
Author(s):  
Moritz Graeff ◽  
Christian S. Hardtke
Keyword(s):  
Terrestrial Ecosystems ◽  
Phloem Transport ◽  
Developmental Trajectory ◽  
Root Tip ◽  
Root Tips ◽  
Arabidopsis Root ◽  
Sieve Elements ◽  
Evolutionary Innovation ◽  
Spatio Temporal ◽  
Cell Niche

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early, so-called protophloem. This is easily observed in Arabidopsis root meristems, where the differentiation of individual protophloem sieve element precursors into interconnected, conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared to protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

scholarly journals Cytokinin-Controlled Gradient Distribution of Auxin in Arabidopsis Root Tip

2021 ◽  
Vol 22 (8) ◽  
pp. 3874
Author(s):  
Lei Wu ◽  
Jun-Li Wang ◽  
Xiao-Feng Li ◽  
Guang-Qin Guo
Keyword(s):  
Growth And Development ◽  
Molecular Mechanisms ◽  
Plant Root ◽  
Primary Root ◽  
Root Tip ◽  
Root Tips ◽  
Arabidopsis Root ◽  
Gradient Distribution ◽  
Shed Light ◽  
Auxin Gradient

The plant root is a dynamic system, which is able to respond promptly to external environmental stimuli by constantly adjusting its growth and development. A key component regulating this growth and development is the finely tuned cross-talk between the auxin and cytokinin phytohormones. The gradient distribution of auxin is not only important for the growth and development of roots, but also for root growth in various response. Recent studies have shed light on the molecular mechanisms of cytokinin-mediated regulation of local auxin biosynthesis/metabolism and redistribution in establishing active auxin gradients, resulting in cell division and differentiation in primary root tips. In this review, we focus our attention on the molecular mechanisms underlying the cytokinin-controlled auxin gradient in root tips.

Histochemical Examination of Invertase Activities in the Growing Tip of the Plant Root

2017 ◽  
Vol 10 (1) ◽  
pp. 35-45
Author(s):  
N.F. Lunkova ◽  
N.A. Burmistrova ◽  
M.S. Krasavina
Keyword(s):  
Plant Root ◽  
Invertase Activity ◽  
Root Tip ◽  
Elongation Zone ◽  
Root Tips ◽  
Phloem Unloading ◽  
Meristem Cell ◽  
Transition To Elongation ◽  
Different Tissues

Background:A growing part of the root is one of the most active sinks for sucrose coming from source leaves through the phloem. In the root, sucrose is unloaded from conducting bundles and is distributed among the surrounding cells. To be involved in the metabolism, sucrose should disintegrate into hexoses by means of degrading enzymes.Aims:The aim of this research was to explore the possibility of the involvement of one such enzymes, invertase, in phloem unloading as well as distribution of its activity in the functionally different tissues of the plant root tips.Method:To estimate the enzyme activities in root tissues, we applied two techniques: the histochemical method using nitro blue tetrazolium. The localization of phloem unloading was studied with carboxyfluorescein, a fluorescent marker for symplastic transport.Results:Invertase activity was not detected in the apical part of the meristem. It appeared only between the basal part of this zone and the beginning of the elongation zone. There is the root phloem unloading in that area. Invertase activity increased with increasing the distance from the root tip and reached the highest values in the region of cell transition to elongation and in the elongation zone. The activities of the enzyme varied in different tissues of the same zone and sometimes in the neighboring cells of the same tissue. Biochemical determination of invertase activity was made in the maize root segments coincident to the zones of meristem, cell elongation and differentiation. The results of both methods of determination of invertase activity were in agreement.Conclusion:It was concluded that phloem unloading correlated with invertase activity, possibly because of the activation of invertase by unloaded sucrose. Invertase is one of the factors involved in the processes preparing the cells for their transition to elongation because the concentration of osmotically active hexoses increases after cleavage of sucrose, that stimulates water entry into the cells, which is necessary for elongation growth.

scholarly journals Effect of earthworms on mycorrhization, root morphology and biomass of silver fir seedlings inoculated with black summer truffle (Tuber aestivum Vittad.)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tina Unuk Nahberger ◽  
Gian Maria Niccolò Benucci ◽  
Hojka Kraigher ◽  
Tine Grebenc
Keyword(s):  
Root System ◽  
Root Biomass ◽  
Fine Root ◽  
Abies Alba ◽  
Fine Root Biomass ◽  
Root Tip ◽  
Silver Fir ◽  
Tuber Aestivum ◽  
Root Tips ◽  
Root Parameters

AbstractSpecies of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.

scholarly journals The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1665
Author(s):  
Natalia Nikonorova ◽  
Evan Murphy ◽  
Cassio Flavio Fonseca de Lima ◽  
Shanshuo Zhu ◽  
Brigitte van de Cotte ◽  
...  
Keyword(s):  
Cell Wall ◽  
Root Growth ◽  
Growth Regulators ◽  
Growth Regulation ◽  
Phosphorylation Site ◽  
Primary Root ◽  
Root Tip ◽  
Arabidopsis Root ◽  
Growth Promoting ◽  
The Impact

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

scholarly journals Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

2003 ◽  
Vol 69 (1) ◽  
pp. 327-333 ◽  
Author(s):  
Renske Landeweert ◽  
Paula Leeflang ◽  
Thom W. Kuyper ◽  
Ellis Hoffland ◽  
Anna Rosling ◽  
...  
Keyword(s):  
Molecular Identification ◽  
Sequence Similarity ◽  
Mineral Soil ◽  
Molecular Techniques ◽  
Root Tip ◽  
Root Tips ◽  
Soil Horizons ◽  
Novel Approach ◽  
Total Dna ◽  
Identification Techniques

ABSTRACT Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.

Proteomic Analyses of Soybean Root Tips During Germination

2014 ◽  
Vol 21 (12) ◽  
pp. 1308-1319
Author(s):  
Setsuko Komatsu ◽  
Myeong W. Oh ◽  
Hee Y. Jang ◽  
Soo J. Kwon ◽  
Hye R. Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Proteomic Analysis ◽  
Plant Root ◽  
Root Tip ◽  
Root Tips ◽  
Form Complex ◽  
Molecular Weights ◽  
Soybean Seedlings ◽  
2D Gel ◽  
Proteomic Techniques

Plant root systems form complex networks with the surrounding soil environment and are controlled by both internal and external factors. To better understand the function of root tips of soybean during germination, three proteomic techniques were used to analyze the protein profiles of root tip cells. Proteins were extracted from the root tips of 4-dayold soybean seedlings and analyzed using two-dimensional (2D) gel electrophoresis-based proteomics, SDS-gel based proteomics, and gel-free proteomics techniques. A total of 121, 862, and 341 proteins were identified in root tips using the 2D gel-based, SDS gel-based, and gel-free proteomic techniques, respectively. The proteins identified by 2D gel-based proteomic analysis were predominantly localized in the cytoplasm, whereas nuclear-localized proteins were most commonly identified by the SDS gel-based and gel-free proteomics techniques. Of the 862 proteins identified in the SDS gelbased proteomic analysis, 190 were protein synthesis-related proteins. Furthermore, 24 proteins identified using the 2Dgel based proteomic technique shifted between acidic and basic isoelectric points, and 2 proteins, heat shock protein 70.2 and AAA-type ATPase, displayed two different molecular weights at the same isoelectric point. Taken together, these results suggest that a number of proteins related to protein synthesis and modification are activated in the root tips of soybean seedlings during germination.

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