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Plant Science

Categories
+ Phycology
+ Plant biochemistry
+ Plant breeding
+ Plant cell biology
+ Plant developmental biology
+ Plant immunity
+ Plant metabolism
+ Plant molecular biology
- Plant physiology
Abiotic stress
Axenic cultivation
Biomineralization
Biotic stress
Endosymbiosis
Ion analysis
Metabolism
Nodulation
Nutrition
Phenotyping
Photosynthesis
Plant growth
- Tissue analysis
Cell wall
Transpiration
Water transport
+ Plant transformation
Protocols in Past Issues

Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach
CC Clément Chambaud
SC Sarah J. Cookson
NO Nathalie Ollat
AB Amélie Bernard
LB Lysiane Brocard
Q&A 0 Q&A
3050 Views
Jan 20, 2023

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana, which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues.


Graphical abstract



Analysis of Monosaccharides from Arabidopsis Seed Mucilage and Whole Seeds Using HPAEC-PAD
GD Gillian H. Dean
KS Kresimir Sola
FU Faride Unda
SM Shawn D. Mansfield
GH George W. Haughn
Q&A 0 Q&A
5814 Views
Dec 20, 2019
Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Tensile Testing Assay for the Measurement of Tissue Stiffness in Arabidopsis Inflorescence Stem
KY Kouki Yoshida
SS Shingo Sakamoto
NM Nobutaka Mitsuda
Q&A 1 Q&A
10228 Views
Aug 5, 2019
Lignocellulosic biomass is a versatile renewable resource for fuels, buildings, crafts, and biomaterials. Strategies of molecularly designing lignocellulose for industrial application has been developed by the discoveries of novel genes after the screenings of various mutants and transformed lines of Arabidopsis whose cell walls could be modified in the inflorescence stem, a model woody tissue. The mechanical properties are used as a quantitative index for the chemorehological behavior of the genetically modified cell wall in the tissue. This parameter can be measured with tensile or bending tests of tissue explants, the vibration analysis of tissue behavior or using atomic force microscopy to probe the tissue surface. Here, we describe in detail the procedure to determine the stiffness of methanol-fixed, rehydrated and pronase-treated inflorescence explants with a tensile testing machine based on classical methods for the determination of cell wall extensibility.

Fluorescein Transport Assay to Assess Bulk Flow of Molecules Through the Hypocotyl in Arabidopsis thaliana
SD Salva Duran-Nebreda
GB George W. Bassel
Q&A 0 Q&A
7577 Views
Apr 5, 2018
The bulk transport of molecules through plant tissues underpins growth and development. The stem acts as a conduit between the upper and low domains of the plant, facilitating transport of solutes and water from the roots to the shoot system, and sugar plus other elaborated metabolites towards the non-photosynthetic organs. In order to perform this function efficiently, the stem needs to be optimized for transport. This is achieved through the formation of vasculature that connects the whole plant but also through connectivity signatures that reduce path length distributions outside the vascular system. This protocol was devised to characterize how cell connectivity affects the bulk flow of molecules traversing the stem. This is achieved by exposing young seedlings to fluorescein, for which no specific transporter is assumed to be present in A. thaliana, and assessing the relative concentration of this fluorescent compound in individual cells of the embryonic stem (hypocotyl) using confocal microscopy and quantitative 3D image analysis after a given exposure time.

Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus
HC Haoxi Chai
YS Yun Shen
Huazhong Shi Huazhong Shi
Q&A 0 Q&A
7874 Views
Aug 5, 2017
Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a polyamine transporter and is able to take up spermidine and its analog paraquat.

Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints
TS Telma E. Scarpeci
MZ María I. Zanor
Estela M. Valle Estela M. Valle
Q&A 2 Q&A
13911 Views
Jun 20, 2017
Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

Measurement of Stomatal Conductance in Rice
YW Yin Wang
TK Toshinori Kinoshita
Q&A 0 Q&A
13804 Views
Apr 20, 2017
Stomatal conductance, the reciprocal of stomatal resistance, represents the gas exchange ability of stomata. Generally, the stomatal conductance is higher when stomata open wider, and vice versa. In this protocol, we describe how to measure stomatal conductance in rice using Li-6400 (Licor, USA).

Simple Methods for Screening and Statistical Analysis of Leaf Epidermal Cells in Dicotyledonous Plants
JT Jian-Jun Tao
SC Shou-Yi Chen
JZ Jin-Song Zhang
Q&A 1 Q&A
13774 Views
Sep 5, 2016
Leaf epidermal cell size and number are positively correlated to leaf area. Stomata are specialized epidermal cells vital for gas exchange and water transpiration. So, observation and statistical analysis of the leaf epidermal cells are valuable for the study of leaf development and response to environmental stimulus. The classical method is using the scanning electron microscope (SEM), which is an expensive and time-consuming method, thus makes the large-scale screening of epidermis impractical. Here we provide simple but effective methods (agarose-based epidermal imprinting and tape-based epidermis tearing) for solving this problem without using the SEM.

Measuring Auxin Transport Capacity in Seedling Roots of Medicago truncatula
Jason Liang Pin Ng Jason Liang Pin Ng
UM Ulrike Mathesius
Q&A 0 Q&A
10131 Views
Jun 20, 2016
Measurement of auxin transport capacity provides quantitative data on the physiological machinery involved in auxin transport within plants. This technique is easy to perform and gives quick results. Radiolabelled auxin (indole-3-acetic-acid) is fed into the roots of Medicago truncatula via an agar block. The resulting radioactivity from radiolabelled auxin uptake in the roots is measured with a liquid scintillation counter. Here, we describe the measurement of auxin transport capacity around the nodulation susceptible zone in young seedling roots of M. truncatula in response to rhizobia inoculation. Similar assays could be adapted in other plant species and to answer other biological questions.

Quantifying Auxin Metabolites in Young Root Tissue of Medicago truncatula by Liquid Chromatography Electrospray-ionisation Quadrupole Time-of-flight (LC-ESI-QTOF) Tandem Mass Spectrometry
Jason Liang Pin Ng Jason Liang Pin Ng
Thy T. Truong Thy T. Truong
Charles H. Hocart Charles H. Hocart
UM Ulrike Mathesius
Q&A 0 Q&A
10675 Views
Jun 20, 2016
Auxins represent a major group of phytohormones controlling plant development. The spatio-temporal regulation of auxin gradients is essential for the initiation, growth and correct development of plant organs. Because auxins and their metabolites occur at trace levels in plant tissue, experiments requiring identification plus their selective and specific quantification can be most conveniently achieved using mass spectrometry (MS) and the associated chromatographic methods. With the advent of appropriate liquid-based ionisation techniques, emphasis has moved from the use of gas chromatography as the sample interface to the MS (GC/MS), with its concomitant need for derivatisation, to the more sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS). We describe an optimized liquid chromatography electrospray-ionisation quadrupole time-of-flight (LC-ESI-QTOF) methodology for the quantification of auxins. While the solvent extraction of young Medicago truncatula (M. truncatula) roots, as described herein, is relatively straightforward, older, woody or oily plant tissues may also be analyzed with appropriate modification to remove interferences and/or enhance extraction efficiency. In our hands, the analytical assay has proved sufficiently sensitive for the quantification of auxins to investigate their roles in various organogenic events, such as root nodulation in M. truncatula. Further increases in sensitivity can be expected with the use of the latest generation of instruments.
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