Hubert Ziegler's research focuses on solute fluxes in plants, their molecular mechanisms,

regulation and functional importance.

For plants, potassium is an essential nutrient. It is taken up from the soil and utilized for growth, development and movement.

Potassium ions contribute significantly to the generation of turgor and in this way drive cell elongation. Following potassium uptake into the root, the cell wall expands and the cell volume increases. Internodes elongate as a result of increasing accumulation with potassium ions.

Leaves develop. In the leaf, turgor-driven microvalves, the stomata, mediate gas exchange between the plant and the atmosphere. As in the root, shoot and leaf cells, potassium uptake into guard cells is mediated by potassium

channel activity. In the following sequence, the strategies and techniques required for the molecular analysis of a plant potassium channel are demonstrated using guard cells from Visscher-Farber. The use of a laser scanning microscope and fluorescent dyes allows clear visualization

of the nucleus. In this organelle, the genetic information for the three-dimensional structure of a potassium channel is stored by nucleic acid sequences or DNA. Following the activation of a particular gene, its DNA sequence is transcribed into messenger RNA and transported to the cytoplasm.

This copy process is called transcription. In the following step, cytoplasmic mRNA is translated into its corresponding amino acid sequence.

The potassium channel protein folds into its three-dimensional structure and inserts into the plasma membrane. The structure and function of a potassium channel reflects the role it plays in a specific cell type, tissue, or developmental stage.

To isolate the gene for a guard cell potassium channel, first the stomata-rich lower epidermis is peeled off the leaf and frozen in liquid nitrogen.

In this way, a picture of the genes which were active before freezing is captured in the mRNA composition of the frozen tissue. The cell walls of guard cells are mechanically robust and resist a turgor pressure of up

to 10 bars. A pestle and mortar are used to break the frozen cells. In the following series of steps, the mRNA will be isolated and purified.

The cell lysate is transferred into a reaction vial and incubated with a denaturing buffer. Under these conditions, the RNA degrading enzymes are inactivated.

Cell walls are separated from the soluble fraction by centrifugation. Under the hood, the resulting supernatant, containing RNA and also DNA and proteins, is treated with phenol chloroform mixture.

The preparation is mixtured and again centrifuged. This organic extraction separates the nucleic acids from the proteins.

In the lower organic phase, accumulation is mainly of proteins. In the interphase of DNA. The water-soluble supernatant is enriched in total RNA. The mRNA content of this fraction is only about 1 to 2%.

The supernatant is transferred to a new vial. Sodium acetate is added.

In the presence of sodium acetate and isopropanol, the now-insoluble RNA precipitates. During centrifugation, order the RNA sediments to the bottom of the reaction vial.

Next, the solubilised total RNA is subjected to an affinity purification process.

The RNA fraction contains mRNA, which is characterised by a chain of adenine nucleotides at the 3' end. This poly-A tail is a structural marker, found neither in ribosomal RNA nor in transfer RNA.

On addition of magnetic beads covered with polythymidine chains, the polyadenine tails of the mRNA form hydrogen bonds with the polythymidines on the magnetic beads.

A strong magnet is used to attract this complex to the side wall of the reaction tube. In this way, the poly-A containing mRNA is separated from the other RNAs.

In the lab, the isolation of mRNA from the total RNA is performed in reaction tubes, which are exposed to a magnet implanted in the sample holder. Non-bound RNA is removed with a pipette.

In the presence of an elution buffer, the mRNA is released from the magnetic bead complex.

It will then be separated electrophoretically in accordance with size and charge. Electrophoresis is performed on an agarose gel. A toothed spacer is used to form pockets in the polymerising gel where the mRNA can later be loaded.

Before loading the mRNA, the two dyes, xylin cyanol and bromphenol blue, are added to the RNA. This mixture of RNA and dye is pipetted into the pockets of the agarose gel. Due to the high density of the glycerol-containing loading buffer,

the RNA mixture sediments to the bottom of the pockets. Total RNA and molecular weight standards are included in the gel as references of known size. On application of a constant voltage, electrophoresis is initiated.

The movement of the two dyes indicates the progressing separation of the still invisible RNA. Within the electrical field, RNAs of different lengths move at different rates, like the differently mobile dyes. After incubation with ethidium bromide, UV light excites RNA fluorescence.

To determine the size distribution of the RNA, the internal molecular weight standards are used. These encompass bands between 9,000 and 200 bases in length. The total RNA is characterised by two prominent bands, the ribosomal 28S and 18S bands.

The mRNA emits diffuse fluorescence in the range between 400 and 4,000 bases.

The identification of the potassium channel gene is accomplished using a DNA copy of the mRNA. This approach allows the several thousand mRNAs, which are normally expressed simultaneously, to be stored as a cDNA library.

Since a large number of genes are always expressed, the isolated fraction of mRNA encodes many proteins of different size and function.

The generation of a cDNA library requires several experimental steps in the lab. These steps will now be demonstrated using a single mRNA molecule as an example. The process of generating a cDNA library begins with a synthesis of a single-stranded cDNA, corresponding to the mRNA.

In the first step, a polythymidine primer is bound to the polyadenine tail of the mRNA. Hydrogen bonds form between the two complementary bases.

Symbols now represent the nucleotides. The mRNA is composed of the nucleotides cytosine, uracil, adenine and guanine.

The reverse transcriptase, which attaches to the poly-T primer, synthesises the corresponding cDNA strand by incorporating single nucleotides from the solution, resulting in an RNA-DNA hybrid.

The synthesis of the first strand is completed. To form the second strand, RNase H is initially required.

It specifically degrades the template mRNA strand.

Next, the activity of a DNA polymerase is required. The gaps created by the RNase H are filled by the DNA polymerase.

All enzymes involved work hand-in-hand. Now a ligase is activated.

The ligase repairs gaps which still exist in the now continuous second DNA strand. All the enzyme reactions which have been shown here one after the other actually take place simultaneously under strict temperature control in a thermocycler.

After purification of the now double-stranded DNA, a ligase is again required. It attaches adapters to the ends of the cDNA, an essential step for the cloning of the DNA.

During this process, single-stranded overhanging ends, or sticky ends, are added to the double-stranded DNA.

In a last step, the individual cDNA strands will be ligated into plasmids which will serve as vectors. Specific restriction enzymes prepare the plasmids for the subsequent cloning of the cDNA.

The free ends of the plasmids now perfectly match the ends of the cDNA. Ligases link the ends to one another. Following these enzyme reactions, each mRNA is represented by a DNA copy at least once in the cDNA library.

Using the functional assay, the cDNA library will now be screened for potassium channel genes. For this purpose, yeast cells are transformed with the plasmids.

Initially, the cDNA plasmid mixture is added to a yeast suspension culture. For the transformation, this suspension is transferred to a cuvette.

Baker's yeast is well suited to screen for a potassium channel gene, since yeast growth, like that of plants, depends on the ability to take up potassium.

The transformation process takes place in an electroporator. Two metal plates attached to the side walls of the cuvette act as capacitor plates across which an electrical field is applied to induce the uptake of the plasmids into the yeast cells.

In response to a short voltage pulse, pores a few nanometers in diameter form in the plasma membrane of the yeast cells. These pores allow the entry of plasmids into the yeast cells.

Some of the incorporated plasmids will contain the potassium channel gene. The transformed yeast cells are now transferred onto a selection medium.

For transformation, a yeast mutant which lacks the ability to take up potassium has been used. This allows the identification of those yeast cells which have received a gene encoding a potassium channel.

During the screening process for functional complementation of yeast growth, transformed cells are plated on medium-low in potassium.

Yeast growth is monitored over several days. A multi-step screen finally identifies yeast strains which grow on media containing minimal potassium concentration and therefore must be functionally expressing a plant potassium channel gene.

A yeast like this will grow, divide and form a colony of several million cells. Growing colonies are isolated and incubated for two to three days in a temperature-controlled chamber.

At the end of the screen, each yeast colony or each clone carries a functional potassium channel gene.

For the following sequence analysis, a large quantity of plasmids is required, so single colonies are transferred from agar plates into liquid culture. To further increase the plasmid yield, an intermediate step is included in which the plasmids are transferred to Escherichia coli.

In the presence of an optimal nutrient supply, the E. coli clone grows in a shaking incubator to produce a dense cell suspension, a basic requirement for the isolation of potassium channel containing plasmids.

The sequence of the potassium channel gene is based on the chain termination method developed by Sanger.

Each of the previously performed sequencing reactions is terminated by a modified form of the bases A, C, G or T. The resulting mixture of different length DNA is now applied to a high-resolution polyacrylamide gel in an automatic sequencer.

In the sequencer, the fluorescently labeled DNA fragments, which differ in length by just a single nucleotide, are separated. A laser scans the gel and detects the fluorescently labeled DNA fragments to determine the sequence of nucleic acids in the potassium channel gene.

The computer assembles the overlapping DNA sequences and unravels the primary structure of the potassium channel gene. The pattern recognized by the laser is digitized by the computer, making it possible to determine the sequence of the bases automatically.

In one scan, the series of bases in the first four A, C, G and T lanes is monitored.

With each new sequencing reaction, the nucleotide chain of the potassium channel gene continuously increases. Eventually, the complete sequence is known. The molecular structure of the channel gene is now identified.

The potassium channel gene was isolated from guard cell mRNA and identified using a yeast complementation assay. It is expressed in the nucleus of the guard cell. After transcription, mRNA is shuttled into the cytosol.

Here, at the ER, ribosomes translate the ribonucleic acid sequence into an amino acid chain. The subunits of the potassium channel are assembled and incorporated into the plasma membrane.

A guard cell is then able to take up potassium ions to regulate guard cell volume and stomatal movement. This process is the result of changes in channel activity and density. In this way, the activity of potassium channel genes and other cell-specific genes

controls plant movement, growth and differentiation, as well as adaptations to changes in their environment. Following the identification of the structure of plant genes, study of the molecular mechanisms regulating these diverse processes is possible.

Modern plant physiology will have a major impact.