The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application

Abstract

Background: The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture from both species.

Results: We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed for small volumes (200 μl). We performed RT-PCR experiments to study the change in the expression of the genes encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (PC5R) in response to abiotic stress.

Conclusions: We found differences between the wheat and barley suspension cultures, barley being more tolerant to the applied osmotic stresses. We suggested a model to explain the obtained differences in stress tolerance between the two species. The suspension cell cultures have proven useful for determining changes in proline concentration and expression level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots.

Figure 1

Chart flow of the initiation and the maintenance of the wheat and barley single cell suspension cultures. Detailed description can be found in the Method section.

Figure 2

Viability and morphology of the wheat and barley cells from the suspension cultures. Zeiss Axioplan 2 fluorescent microscope was used for visualising the fluorescein diacetate (FDA) treated cells. Data are the means of four independent experiments, and bars represent ± SE; n = 4.

Figure 3

Production of fluorescein by wheat suspension culture. Effect of the fluorescein diacetate (FDA) concentration on the uptake and production of fluorescein by wheat cells.

Figure 4

The predicted P5CR protein sequence comparison from wheat and barley. The wheat protein sequence is from GenBank (Triticum aestivum GenBank:AAW82908) and Hordeum vulgare GenBank:AY177684). The comparison was performed with CLUSTAL W (1.83) multiple sequence alignment program. The highlighted region is the peptide, which was used for the antibody production.

Figure 5

Western blots of the suspension culture. P5CR protein identification from the salt soluble fraction of the suspension cultures were performed by immuno blotting: with polyclonal P5CR peptide antibody. MW: high protein molecular weight marker (Invitrogen Inc); B: barley and W: wheat.

Figure 6

Changes in cell viability under osmotic stresses. Cell viability was expressed as a percentage of the number of cells that remained alive in media supplemented with 50 mM NaCl, 100 mM NaCl, or 5% PEG 6000 with respect to time. A) Wheat (PC998) B) Barley (PC1163). Data are the means of three independent experiments, and bars represent ± SE; n = 3. Values marked with different letters are significantly different from the respective control at P <0.05 as detected by a repeated measures ANOVA.

Figure 7

Changes in proline content of the cells under osmotic stresses. Proline content of live cells in media supplemented with 50 m M NaCl, 100 mM NaCl, or 5% PEG with respect to time. A) Wheat (PC998); B) Barley (PC1163). Data are the means of three independent experiments, and bars represent ± SE; n = 3. Values marked with different letters are significantly different from the respective control at P <0.05 as detected by a repeated measures ANOVA.

Figure 8

Wheat gene expression studies under osmotic stresses. The RT-PCR expression analysis of the genes encoding wheat P5CS and P5CR were performed from cells subject to abiotic stress with respect to time. The graphs were generated by normalizing gene of interest expression against tubulin using the image analysis software ImageJ.

Figure 9

Barley gene expression studies under osmotic stresses. The RT-PCR expression analysis of the genes encoding barley P5CS and P5CR from cells subject to abiotic stress with respect to time. The graphs were generated by normalizing expression of gene of interest against tubulin using the image analysis software ImageJ.

Figure 10

The proposed model to explain the obtained differences in stress tolerance between wheat and barley suspension culture. Diagrammatic model proposed to integrate the results obtained with the those reported in the literature to account for the differential response of wheat and barley to abiotic stress: For wheat under non-stressed conditions the high steady state level of proline is maintained by low rate of turnover/recycling activity of Pox, the net result is proline levels in wheat are close to a hypothetical 'threshold'. While in barley Pox activity is higher resulting in lower steady state levels of proline, which is below the 'threshold'. Under abiotic stress the activity of Pox is reduced and P5CS and P5CR genes are up regulated and proline increases in both wheat and barley, although for barley the proline level remains below the 'threshold and therefore does not suffer proline toxicity and tolerates the stress more efficiently. The colour intensity and thickness of the arrows represent the expression level differences in the different part of the pathways.