Prev Next
Research

Methods

17 June, 2013

High Resolution Plant Phenotyping Platform

Contents

Immunolocalization

Our immunofluorescence protocol can be used to assay sub cellular localization of proteins and DNA in plants at all stages of development, from seed germination to embryo formation.

The protocol includes the following procedures: fixation, tissue cleaning, partially digestion of cell wall, permeabilization and immunostaining of samples. It includes the description of the key steps, guidelines for troubleshooting and examples of data obtained using these methods. Due to the good tissue preservation this protocol allows to perform three-dimensional reconstruction of the cellular organization of entire plant organs after imaging.

The method significantly improves the accuracy of protein expression - and localization studies compared with older protocols. Overall, this protocol in its shortest version takes up to 5 hours to complete and includes only few steps. This might make it applicable for a robotized handling with very low antibody volume up to 50 μl. It is suitable for a wide range of plant species and different organs and cells types. The robotized platform has been developed also for immunolocalization in the single cells, including protoplasts and suspension cells.

A novel high efficiency, low cost, fast system for growth, flowering and in vitro seeds formation of Arabidopsis thaliana

Abstract

An in vitro system has been designed for the growth and characterization of the Arabidopsis thaliana plants with further seeds formation. The system consists from two steps:

  1. Plants cultivation on standard medium that allow to perform selection steps and lines characterization, including investigations of gene expression by immunolocalization and RNA analysis.
  2. Formation of the sterile seeds on Hoagland medium. The system allows us to perform cycle from seeds to seeds about 45 days. This can significantly accelerate time for obtaining pure homozygotic lines after crossing or transformation. The sterility during all steps of plant growth till seeds drying allows us to prevent any contaminations coming in greenhouse from cross-pollination and omit further seeds sterilization step. In addition, we are able to significantly reduce the amount of the waste generated from the soil. In the wild type we are able to generate more than 350 seeds per flowering stem/per plants, while in the case of the severe mutants the amount of seeds were restricted to 100 per tube.

In addition, the system allows us to overcome experimental problems with majority of the severe mutants, mainly with defect in root development. The system also has been applied for generation of N15 seeds, which have been used for Stable isotope labeling with amino acids in cell culture (SILAC). In our hand we were able to generate seeds which have more than 98% of N15 contents. As examples of system application, homozygotic seeds of 2 different mutants have been generated:

  • Rootless GSH-deficient rml1 with detect in auxin response
  • PLETHORA (PLT) triple mutant with severe defect in cell polarity and stem cell formation in the root.

Conclusions

The system described here allows us to drive small and large scale cultures of Arabidopsis plants. It's major advantages are flexibility for any Arabidopsis ecotype and mutants, including one lacking root systems, easy handling, fast seeds formation and low cost. The protocol is suitable for many experimental purposes.

Comparison of different methods of seeds generation in Arabidopsis.

Parameters

Methods

Standard in soil

In vitro

Space per plant

30 sq.sm

2 sq.cm

Duration from seeds to seeds

60 -70 days

40-45 days

Amount of waste

300 ml

3 ml

Seeds per plants

1500

400

Genetic seeds “purity”

Moderate, special covering require

100 % purity

Watering

Yes

No

Mutants with defect in development

Very low yield, not applicable for the plants without roots

Applicable to the mutant without roots (rml1, plt1,2,3).

Generation of seeds containing special labeling (N15 seeds)

Not applicable

Applicable

Root  characterization and primary molecular analysis during growth

Not applicable

Applicable, including immunolocalization

All times are counted from seeds planting point.

Methods of the investigation of the gene expression in plants: an overview.

{% blockquote %} "All animals are equal, but some animals are more equal than others." - George Orwel {% endblockquote %}

Our knowledge’s about gene expression is a key for the understanding of the mechanism of the biological process. That's why it is very important to choose a reliable methodology for investigation for gene expression at the cellular level and its quantification. Gene expression includes at least 3 level of the regulation:

  1. Transcriptional level (mRNA)
  2. Translational level (protein)
  3. Post-transcriptional level: formation of the functional protein clusters/complexes.

All these 3 levels are require appropriate methods, what, indeed, dependent from the object of the investigation. Generally, object of the investigation can be separate to 2 main groups: multicellular organism with highly hetero-genus cell types and homogenous cell type like cultured in vitro cell.

From this point of view, the objects with heterogeneous cell population do not allow to use majority of the modern molecular methods. The main pitfall is the quantification of the mRNA/protein level.

This is very easy in the case of homogenous cell population, but became much more complicated in the case of the multicellular organism. In the last case all cell types and even cell of the same type, but with different spatial position have a n different chromatin organization, that's why different regulation of the gene expression at mRNA level. In addition, dependence from the presence of the protein-partners, even neighbor cell with the same cell fate, may have different protein level, and protein activity level. In addition, in majority of the cases alteration in the mRNA and protein level occurred in different direction in different part of the multicellular organism in response to different stimuli. It may mean that average changes in mRNA/protein level may not reflect real changes in the key cells/organs.

Below we describe methods which are suitable for quantification of the gene expression in the case of multicellular organism and in the case of the homogenous cell population.

Transcriptional level:

For the transcriptional level the most popular method is microarray, what have bene used in numerous investigation. However, this method cannot be applied to heterogeneous cell population, because they do not give even answer about alteration in the trends of gene expression. Namely, in many case specific mRNA

Translational level:

For the transcriptional regulation, western blot serve as a very popular tool for determination of the protein level. The conclusion has been usually made on the intensity of the bands on the WB membrane. The modern detection method allows researcher measure intensity pf the chemiluminsecsce very precisely. However, surprisingly, even such precise measure of the band intensity did not give precise information about the protein level! Why? The main problem is the way of normalization. Usually normalization was done per equal amount of the total protein isolated from whole organism (like from whole Arabidopsis plants, for example). The results of such normalization represent the average between cells with different protein level and which changes in many cases in opposite direction. But, according to basic rules of the statistics average have sense only in the case if changes occurred in one direction only.

Post-transcriptional level:

In majority of the cases protein functions have been regulated through interaction of the proteins with partners and formation of the protein-protein complex. There are two different level of the investigation of the protein communication: In vitro and in vivo assays. Protein co-immnuno-precipitation allow researchers to investigate protein complex after isolation. Unfortunately, there are quite a lot possible artifacts which require additional proofs. In order to minimize any artifacts related with co-immuno precipitation assay and keep conditions as close to reality as possible, PLA analysis has been applied for investigation of the proteins complex stability. This assay offers us the ability to survey protein complex investigation directly in Arabidopsis roots, detecting targets in their natural unperturbed environment.

{% blockquote %} "All cells in organism are equal for gene contents (DNA), but all of the cells are different in the term of gene expression (RNA and protein activity)” {% endblockquote %}

Investigation of the gene expression in plants.

During last decade a great progress has been achieved in the methodology of investigation of gene expression in eukaryotes (both plants and animals). New methods have been developed to study gene expression at different level: microarray for RNA level, proteomics for proteins and its activity levels and finally, metabolomics for investigation of metabolism. Moreover, new improvements to the old technique like real-time PCR and WB are also available, which is significantly increasing sensitivity and accuracy of the quantifications. Surprisingly, in many cases usage of these methods is associated with some confusion. The main reason for that might be a fact that multicellular organism consist of different cell types at various developmental stages, which express totally different set of genes and activate different metabolic pathway in response to stimuli. Even such simple system as roots of higher plants has different zones with different metabolomics in each. In many cases even neighboring cells express completely different proteins.

It means that majority of the “new-old methods” cannot be applied for a whole organism, but have a restrictive usage only to homogenous cell populations.

New technologies for microscopy, and new tools for in situ RNA and protein localizations (in situ and immunolocalization), can aid to find a solution for this problem. A crucial step is a separation of various cell files of multicellular organism and study gene expression in each cell types and each cell individually.

However, so far majority of the protocol for in situ localization lack coordinate system inside organs, precise position of the proteins in the organs and inside cells in 3 dimensions, despite of the fact that current microscopy techniques allow researchers to perform precise high quality 3D reconstruction.

In order to determine precisely position a protein in the tissue on a level of a cellular resolution it is necessary to distinguish a cell border and nucleus as hallmarks for each cell. There are at least three possible protocols to perform it.

Cell membrane can be chosen as a marker. In this approach a EGFP-LTI6b line can be used however it requires a crossing of lines of interest with this marker line as well as introduction of a fluorescent protein. To overcome this direct labeling of either a cell wall or plasma membrane as post-immunolocalization treatment can be used.

Approach 1: Plasma membrane staining.

FM4-64 (FM1-43) is a lipophilic cationic dye which interacts with charged membrane phospholipids. This dye is very useful for the living cells under control conditions. Usage under stress conditions can lead to internalization of this dye.

Plasma membrane is a very dynamic border, which is very sensitive to environment. In the living not-perturbing conditions PM is a good marker for the living cells, but after fixation (which requires for majority of the in situ and immunolocalization procedure, PM loose attractive properties for the cationic lipophilic dyes).

The analog FM4-64 FX has a limiting capability to keep localization on the plasma membrane even after middle fixation. But this dye rather incompatible with protein immunolocalization procedure which requires severe membrane permeabilisation for antibody penetration. Altogether, plasma membrane is not an excellent tool for cell border markers in plant cells, especially in the combination with protein localization. The other approach is to use a fluorescent protein as membrane marker (Kurup et al., 2005). However, such approach require extensive efforts in order to introduce marker to mutant lines and in many case do not compatible with protein localization.

Approach 2 (Truernit et al., 2008): Polysaccharide staining with propidium iodine.

The method is based on ability of propidium iodine to bund to polysaccharide after its oxidation by periodic acid and opening of polysaccharides bonds. Dye (PI in this case) can bind to these open bonds in the presence of sodium bisulfite and hydrochloric acid. This binding can be observed only at final pH of the staining solution of around 1.5-2 pH units, which is unfortunately extreme low and does not allow nucleus (DNA) staining. This method of cell border identification cannot be combined with protein localization, but can be combined only with GUS reaction and other histochemical techniques, like ROS measurements. However, protein and DNA detection is not possible with this approach due to very low pH of the mounting solution. pH 7 is required for protein and nucleic acid detection.

Approach 3: Cell wall staining with calcofluor white, protein detection with antibodies and nucleus detection with propidium iodine.

This approach is most suitable for majority of the applications, including protein localization, cell cycle investigation etc. In addition, last technique can be very useful for detection of the cell wall thickness/composition.

Protocol for double labeling.

  • Fixation: 40-45 minutes in 2% FA.
  • Washing 1: H2O, 2 minutes
  • Cuticle dissolving: warm methanol, 60°C, 30 minutes, 2 times total.
  • Re-hydration: gradual addition of the water, 20 minutes.
  • Cell wall digestion: 5-7 minutes, 0.25% Dricelaze, 0.1 % Macerozyme. Reason: to make cell wall more uniform through tissues and increasing enzyme permeability for RNAse.
  • Washing: 2 minutes, 25 mM Tris, pH 7.5
  • RNA digestion: 60-90 minutes, 37 C in 25 mM Tris, pH 7,5, 2 U/ml. Aim: to remove RNA because PI can bound to RNA as well.
  • Washing: 2x 25 mM Tris, pH 7.5.
  • PI staining: 4 mg/l PI in 25 mM TRis, pH 7.5, 20 minutes.
  • Washing: 1x 25 mM Tris, pH 7.5: 1x 25 mM Tris, pH 9.5. Reason: to change pH to alkaline for preventing BR28 precipitation and for good cellulose binding.
  • BR staining: 10 mg/l BR28 in 25 mM Tris, pH 9.5, 20 minutes.
  • Washing 2x in 25 mM Tris, pH 9.5, 5 minutes.
  • Mounting: solution: 40 µl S2838 + 40 µl 50% Glycerol + 40 µl 500 mM Tris, pH 9.5.
  • Scaning: BR28: 365-405 or 2P laser; PI: 543-61 or 2P laser.

Total timing of the procedure: 4-5 hours (if no protein localization). In the case of protein localization time will increase to maximal 8 hours.

Troubleshooting in the case of the roots:

Intense staining in the outer cell layers, especially in the elongation zone, but very low relative staining in the stele of the RAM Reason: primary and secondary cell wall have a different thickness and composition. To make such thickness more equal, one need to adjust cellulose digestion enzyme composition and duration. PI staining have a diffuse labeling around nucleus. Reason: incomplete RNA digestion. Decision: increase RNA incubation time. BR28 has a diffuse labeling inside cell. Reason: BR28 precipitation due to low pH. Decision: increasing washing time in the buffer with pH 9.5. In our hand no any problem with leaf staining has been observed. However, the same problems as for the RAM due to different cell wall density may exist for staining of the SAM region due to big variation in cell wall properties.

Conclusion

Here we have presented a reliable method which allows us to precisely perform proteins localization with layers and cells resolutions. This method open many new approaches for understanding the gene function in the plant contents.

Anticipated examples

Figure 1. Abnormalities in root meristem have been detected after cell wall/nucleus staining and 3D reconstruction. Figure 2. 3D reconstruction of the leaf development.

Literature

Kurup, S., Runions, J., Köhler, U., Laplaze, L., Hodge, S. and Haseloff, J. (2005)
Marking cell lineages in living tissues.
The Plant Journal, 42: 444–453.

Truernit, E., Bauby, H., Dubreucq, B., Grandjean, O., Runions, J., Barthélémy, J., & Palauqui, J. C. (2008)
High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis.
The Plant Cell Online, 20(6): 1494-1503.

Pollen embryogenesis and its application in plant breeding and biotechnology.

The production of doubled haploids has become an important tool in advanced plant breeding institutes and commercial companies for breeding crop species breeding. Isolated pollen serves as an ideal source for the haplod production. The switch of gametophytic program to pollen embryogenesis with the further formation of the homozygous plants for production of new isogenic lines has a fundamental importance for plant breeding. This switch has been induced by stress like heat stress etc. However, molecular mechanism of such switch is unclear and no specific genes responsible for pollen embryogenesis have been detected so far.

Here we proposed hypothesis that switching of developmental program towards pollen embryogenesis is a part of general pathway of somatic cell re-programming and regulated epigenetically through regulation of the chromatin structure. That’s why numerous attempts to find specific genes for pollen embryogenesis were unsuccessful.

Chromatin dynamics is a key epigenetic regulator of cell re-programming from differentiated somatic to dividing and especially to totipotent one. From this point of view transition of somatic cell to embryogenic (totipotent) stage can be dividing in 2 different steps (stages) which characterized by different chromatin dynamics. At the first stage cell which have a defined fate in planta (mesophyll cells, pollen etc.) should reactivate cell cycle. However, in planta cells which undergo rapid cell division already establishes cell fate and cannot be considering as totipotent without transition to de-differentiated stage. Correspondingly, only stem cells can be considered as totipotent. That’s why second step, ea. transition from dividing cell to totipotent represent a key step for realization of the totipotency program. The key differences between dividing and totipotent cells is a chromatin dynamics, which regulated rather epigenetically through HDAC activity and directly dependent from key plant phytohormone auxin. The fact that majority of the stress-responses in plants were regulated epigenetically fit well with the fact that stress is necessary for induction of totipotency as second step in cell re-programming (Chinnusamy, V., & Zhu, J. K. 2009).

Pollen after meiosis is a relatively cell-cycle active cells which in planta undergo to maturation (partial differentiation) with further significant vacuolar growth (pollen tubes growth). However, under certain experimental condition one can prevent further pollen differentiation and switch the program to cell cycle activity. This process is named “nuclear reprogramming” (Gurdon and Melton, 2008). “Nuclear reprogramming” can most simply be defined as a process of alteration in the nucleus structure (chromatin dynamics).

In the case of the pollen such switching and cell de-differentiation (“nuclear re-programming”) were accompanied by rapid changes in the chromatin structure (Testillano, P. S., et al., 2000). In addition, it has been recently shown such changes in nucleus organization during pollen embryogenesis were regulated by DNA methylation (El-Tantawy, A. A.et al., 2014).

Chromatin structure (nucleus shape, density, and even in more extension, nucleolus function) is an excellent marker of cell re-programming from somatic (pollen) to embryogenesis. As it has been shown previously (Schmidt et al., 2014), cell in the de-differentiated stage in planta were characterized by extremely specific nucleus shape (round with very small size) and a very small nucleolus. These features can be easy determined by double DAPI/PI labeling (ref).

So far there is only one factor which is known to induce de-differentiation: auxin under high initial internal accumulation. It is also well-known that stress factors like high temperature (33° C), and even in more extension ASA and lactones, serve as an ideal factor to induced cell de- differentiation. The involvement of the auxin in pollen development have been pointed by high activity of the auxin biosynthesis genes YUC2 and YUC6 during pollen development, as well as activity of TIR1 and AFB1,2,3 at the stage after meiosis (Cecchetti, V. et al., 2008). The next possible factor what regulate pollen embryogenesis is auxin transporter PIN8. Loss of PIN8 function prevents pollen maturation and may led to switching of the pollen to hametophytic pathway.

The involvement of the chromatin structure alteration in the pollen re-programming to embryogenic pathway has been confirmed in the recent investigation as well (Li. H et al., 2014).

Protoplasts as versatile systems for investigation of the plant cell reprogramming

Plants are sessile organisms and have remarkable developmental plasticity ensuring their permanent adaptation to the environmental ques. An extreme case of adaptation process is plant cell totipotence what may realize as plant regeneration from somatic cells (somatic embryogenesis) in response to exogenous and/or endogenous signals. Single cell population (protoplasts) is a suitable system for investigation of molecular mechanism of totipotence realization. In this review we overview the mechanism that can lead to initiation of cell division in single differentiated somatic plant cells. Four different steps in protoplasts re-programming have ben described with focus on epigenetic, genetic and physiological characterization of each of them. We highlighted the importance of initial explant metabolic status and isolation procedure as per-request to cell re-programming. We strength importance of the auxin signaling and it’s interaction with stress-regulated pathways during the induction cell cycle and further totipotence induction and realization. Despite several reviews have been published last time in the topic, system biology of the plant cell re-programming has not been described.

Cells of higher plants can retain their regenerative potentialities whencultured in vitro. Some cell types are able to regenerate organs or even whole plantlets through organogenesis or somatic embryogenesis. Yet only some of the cell types in certain developmental stages (especially in monocotyledon plants) capable to re-enter cell division cycle, but regenerative capacity are not stable and are usually lost rapidly after isolation of the tissues. Moreover, even in dicotyledonous plants capability of plant regeneration is strictly dependent from a genotype and an initial status of the cell. The causes of this phenomenon are unknown and can’t be explained from a genetic point of view: in planta all genotype have similar meristems and able to perform zygotic embryogenesis. Recent investigations pointed out the importance of epigenetics (alteration in chromatin structure/accessibility) in regulation of both cell differentiation and de-differentiation (Shemer, O., et al., 2015; Sugimoto K. et al., 2019; Pasternak and Dúdits, 2019). In studies of phenomenon of plant cell reprogramming and its mechanisms, different systems are used based mainly on leaf or root multicellular explants. We believe, however, that plant protoplasts is a versatile system for study these processes and have a number of significant benefits. So we will describe here these advantages of protoplasts, paying attention to changes in chromatin status during all stages of re-programming. First, we decided to describe in details all steps in cell programming during embryogenesis and post-embryogenic development, This is particularly important because cell de-differentiation is a process opposite to differentiation and should pass opposite steps.

Literature

Cheng Y.,Dai X., Zhao Y. (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. 20, 1790–1799

Cecchetti, V., Altamura, M. M., Falasca, G., Costantino, P., & Cardarelli, M. (2008). Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation.
The Plant Cell Online, 20(7), 1760-1774

Chinnusamy, V., & Zhu, J. K. (2009).
Epigenetic regulation of stress responses in plants.
Current opinion in plant biology, 12(2), 133-139

El-Tantawy, A. A., Solís, M. T., Risueño, M. C., & Testillano, P. S. (2014)
Changes in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley.
Cytogenet Genome Res, 143, 200-208

Feher, A., Pasternak, T. P., & Dudits, D. (2003)
Transition of somatic plant cells to an embryogenic state.
Plant Cell, Tissue and Organ Culture, 74(3), 201-228 link

Gurdon J. B., Melton D. A. (2008)
Nuclear reprogramming in cells.
Science 322, 1811-1815

Li, H., Soriano, M., Cordewener, J., Muiño, J. M., Riksen, T., Fukuoka, H., ... & Boutilier, K. (2014)
The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte.
The Plant Cell Online, tpc-113

Sakata, T., Oshino, T., Miura, S., Tomabechi, M., Tsunaga, Y., Higashitani, N., ... & Higashitani, A. (2010)
Auxin reverse plant male sterility caused by high temperatures.
Proceedings of the National Academy of Sciences, 107(19), 8569-8574

Schmidt, T., Pasternak, T., Liu, K., Blein, T., Aubry-Hivet, D., Dovzhenko, A., ... & Palme, K. (2014)
The iRoCS Toolbox–3D Analysis of the Plant Root Apical Meristem at Cellular Resolution.
The Plant Journal. 77 (5), 806-814

Testillano, P. S., Coronado, M. J., Seguí, J. M., Domenech, J., Gonzalez-Melendi, P., Raška, I., & Risueno, M. C. (2000)
Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis.
Journal of structural biology, 129(2), 223-232

More coming soon...

The excellent robotic system to perform immunolocalization have been made by Intavis.