Biochemistry of the Multiple Forms of Glycosidases in Plants

We have been investigating an electrochemical single-molecule counting experiment called nanopore resistive-pulse sensing. The sensor element is a conically shaped gold nanotube embedded in a thin polymeric membrane. We have been especially interested in counting protein molecules using these nanotube sensors. This is accomplished by placing the nanotube membrane between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ionic current flowing through the nanopore. In simplest terms, when a protein molecule enters and translocates the nanopore, it transiently blocks the ion current, resulting in a downward current pulse. We have found that the duration of such current-pulses are many orders of magnitude longer than the electrophoretic transport time of the protein through the nanotube detection zone. We develop here a simple model that accounts for this key, and previously explained, observation. This model assumes that the protein molecule engages in repeated adsorption/desorption events to/from the nanotube walls as it translocates through the detection zone. This model not only accounts for the long pulse duration but also for the triangular shape of the current pulse and the increase in the standard deviation of the pulse duration with increasing protein size. Furthermore, the results of our analyses are in general agreement with results obtained from other investigations of protein adsorption to surfaces. This includes the observations that smaller proteins stick more readily to the surface but remain adsorbed for shorter times than larger proteins. In addition, the sticking probabilities calculated from our data are in general agreement with results obtained from other methods.

An adsorption-based model for pulse duration in resistive-pulse protein sensing.

Publisher Summary This chapter investigates the pollen transformation, or better transformation using pollen as “supervectors.” Using this method, swelling and germinating pollen are incubated with exogenous gene material and then used for pollination and fertilization of the species from which the pollen is collected. This is a much simpler method to use than other gene transfer techniques, as it avoids somaclonal variation, and, it should work with cereals. Microgametophytes are used to transfer exogenous DNA, either as total DNA or within a plasmid or phage vector. The transformation experiments described are based on direct gene transfers. Efficiency of coculturing Agrobacteria with regenerating protoplasts, however, is much higher than in direct transfers. Another method to take advantage of natural fertilization processes in transformation experiments is the introduction of donor DNA into ovaries at approximately the time of fertilization. Three possibilities that need to be considered for transferring the exogenous DNA into the new generation are—(1) the pollen tube, just arrived near the ovules, could take up the DNA and transfer it into the egg cell; (2) the egg cell could take up the DNA; and (3) the DNA could be taken up immediately after fertilization either into the zygote or the developing embryo.

Pollen-Based Techniques in Genetic Manipulation

Utilization of Different Sugars as Carbon Source for in vitro Anther Culture of Petunia

Using the Arrhenius thermodynamic equation, which relates rates of processes to temperature through the quantity E

 a
 , the ‘Heat (or Energy) of Activation’, we have evaluated the thermal relationships for several parameters of Hg cycling. It is shown that release from isolated leaves (shoots) of Hg0 is a two-step process with a higher E

 a
 value below 21 °C than above (28 vs 14 kcal mol−1). Open field air Hg measurements over a mixed stand of grasses and other plants in volcanic soil show strikingly similar behavior to detached organs. Mercury release from volcanic soil was uniform over a wide temperature range, resembling plant and open field emissions above 18 to 21°C with anE

 a
 value of 13 kcal · mol−1. We conclude that Hg release below 18 to 21 °C is limited by a physiological process, whereas above that range, release is controlled by the physical process of vaporization itself. Plant concentration of total Hg from 5 to 33 °C (air temperature), is a more complex function involving direct accumulation and re-release of Hg0 from the atmosphere, release from tissue storage, and root uptake with reduction.

Marlene Komp

Papers

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

β-Galactosidase from Petunia hybrida: characterization and differentiation from E. coli β-galactosidase activity