ReviewHigh resolution spatial analysis of plant systems
Introduction
Specific physiological conditions prevail at every morphological level within an organism (i.e. at entirety, organ, tissue, and cell levels). Analysis of the chemical composition of an organism on a supra-cellular scale provides only limited information, as the measured results represent an average for everything included in the sample. To understand the physiology of a whole organism, it is necessary to understand the behaviour of a single cell as its fundamental unit. Information garnered from the analysis of samples extracted at high resolution promises to advance the knowledge of such diverse processes as cell differentiation, signal transduction, and physiological responses to external stimuli such as toxins or pathogens. Hence, the ability to generate samples at high resolution is of outstanding interest, especially to those researching disease generation, avoidance, and control. For this reason, research on single cells is rather advanced in the animal (and especially human) sciences. In comparison, research on single cells from higher plants is relatively difficult, mainly because of their small size and rigid cell walls, and has been more or less neglected so far.
The major focuses of the high-resolution analysis of both animal and plant tissues are the determination of local patterns of gene expression, protein composition, and solute concentrations. In plants especially, advances require the development of suitable methods for the sampling, handling, and qualitative and quantitative analysis of minute amounts of tissue. There are three practicable routes to overcoming the problem of starting with a limited amount of material. First, the development of assay systems that can cope with small volumes; second, the development of assays that are sufficiently sensitive to handle the extremely low concentrations caused by dilution; and third, the generation of techniques that allow the collection of many cells from the specific cell type of interest.
In this review, I summarise the current state of techniques for sampling and analysing plant tissues at high spatial resolution and the recent advances in the animal field, and I discuss the potential for transferring animal-derived methods to plant systems.
Section snippets
Sampling single cells
In plants as well as in animals, samples can be extracted from a living organism, isolated cells, or sections of fixed tissue. Compared to sampling from living objects, the embedding of samples obviously disturbs the dynamic behaviour of the tissue and might result in the loss of information relating to transient events. Measurements in a living system are desirable for the determination of concentration changes and metabolite fluxes over time or in response to altered environmental conditions.
Analysis of gene expression
Following recent advances in the genome sequencing of animals and plants, the current challenge is to unravel the interactions among gene products on all morphological levels. Although knowledge of mRNA patterns does not directly address gene function, information about the temporal and spatial expression pattern of a gene can provide valuable insights into its potential role. In situ hybridisation or in situ reverse transcriptase polymerase chain reaction (RT-PCR) 7•• in fixed tissue sections
Analysis of proteins
Proteomics-based studies at the level of tissues or single cells offer a powerful complementary approach to RNA-expression profiling. As proteins catalyse most reactions in a cell, the structure of a protein can provide important information about the function of the gene that encodes it. The analysis of proteins from small samples is not a well-established biochemical technique, probably because the protein concentration of samples is normally rather low and there are no amplification
Analysis of solutes
In extracted picolitre samples, small molecules such as sugars or inorganic ions can be quantified by microfluorometric enzyme assays 21 or energy dispersive X-ray microanalysis, respectively 2. Unfortunately, these analytical methods include no substance separation. Therefore, they lack the potential to quantify a variety of compounds simultaneously, moreover, they are prone to interfering interactions among sample components. Separation systems such as capillary electrophoresis (CE) seem to
Physical methods
Although this review emphasises the analysis of macromolecules and solutes, the existence of additional techniques for the determination of physical cell para-meters, such as turgor pressure, or membrane and water potential, in single plant cells should be mentioned 1•, 33•. Such measurements are mostly restricted to surface cells but in some plant organs, for example, in roots, turgor pressure gradients among non-surface cells could also be determined 34. In addition, growth rates 35 and
Conclusions
Most of the biochemical techniques for high-resolution spatial analysis described here originate from the medical field and, so far, have been used rather infrequently in plants. In principle, all of the techniques available for animal cells should also be applicable to plant systems with minor or major modifications. I hope that this review might encourage plant scientists to adapt methods used in medical research.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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