The role of transport proteins in eukaryotic organisms and their potential exploitation in genetically modified plants There are three major types of membrane transport proteins (Lodish, et. al, 1995). ATP-powered pumps derive the energy required for energetically unfavorable transport of ions or molecules via the hydrolysis of ATP. Channel proteins engage in passive transport, moving particular ions, or water down their respective concentration gradients. Transporters use the slowest mechanism for transport binding only one or a few substrate molecules for transport at a time. All three of these types of molecules contribute to the amazing selectivity of plasma membranes and are, thus, critical to the organization and function of the entire cell. ATP-powered pumps, also referred to as ATPases given their apparently enzymatic properties, catalyze the energetically unfavorable movement of ions against their concentration gradient. This type of transport protein is further subdivided into three classes, namely P, V, and F.
Common to all three types of ATP-powered pumps are the one or more binding sites for ATP on the cystolic side of the membrane. Made up of four polypeptides, two alpha and two beta peptides, P-class pumps are the only class that become phosphorylated as part of the transport cycle. F and V class pumps are similar in structure, both having multiple transmembrane proteins and an extrinsic group of least five kinds of polypeptides called the cytosolic domain. Both do not require phosphorylation to be active, but F and V ATPases differ in their functions. Where V-pumps maintain hydrogen ion gradients across membranes by using ATP, F-pumps primarily serve to make ATP from ADP and phosphate ions.
Every channel proteins can be grouped into one of two classes by examining its functionality. Some channels are continuously open. Other transmembrane channels require a message before they will open to allow for the flow of ions. A fast rate of transport characterizes either type of this group of proteins. Transporters use a much slower mechanism to transport ions and molecules.
In this case, either one or a few ions or molecules affix themselves to the transport protein. The binding of the substrates causes a conformational change such that the ions or molecules of interest are exchanged from one side of the cell to the other. The many types of transporters are sub-divided into classes according to the number of substrates that the protein binds and the relative direction of transport of substrates for transporters that bind more than one substrate. Uniporters bind and transport one substrate at a time. Two or more ligands attach to the same face of a symporter; the symporter subsequently transports both ligands in the same direction. Antiporters are similar to symporters in that they bind multiple ligands, but the substrates bound to an antiporter are transported in opposite directions relative to each other.
In symporters and antiporters one bound molecule or ion tends to move along its concentration gradient, while the others move in an energetically unfavorable direction. A completely different type transmembrane protein, namely bacteriorhodopsin (Lodish, 1995), which uses light energy to cause the conformational change required for transport, characterizes lesion-mimic phenomena in plants. Plants are actually susceptible only to a select and small number of pathogens. The majority of pathogens are incompatible with plants, but despite incompatible pathogens imposing no actual threat to plants, when a plant recognizes an incompatible pathogen, it responds with apparently drastic measures. Plants trigger a cell death pathway, which is activated by the expression of a bacterial proton pump in tobacco (Mittler, Shulaev & Lam, 1995). This pump, the bO pump, serves to transport hydrogen ions and is activated when the protein’s retinal group absorbs light. Spontaneous lesions, resembling hypersensitive response lesions, form with bO pump activity.
Several protective mechanisms switch on at this point and systemic acquired resistance manifests itself. Synthesis of pathogenesis-related proteins increases, as does the volume of salicylic acid stored. Despite the pathogen being incompatible, the cell reacts as if an actual threatening pathogen was present. Earlier research investigated the more general topic of systemic acquired resistance, a phenomenon that is the result of bO pump activity (Hebers et al., 1996). Systemic acquired resistance brings about immunity in unaffected parts of the plant that shows lesion-mimic activity elsewhere. More recent studies have taken this concept and sought the fundamental causes and initiators of systemic acquired resistance.
How might this phenomenon of the host’s defense mechanisms being activated in the absence of a pathogen be manipulated and applied to better agricultural crops? Constitutive expression of the H. halobium bO gene could theoretically confers resistance to pathogens that would usually threaten important crop plants such as the potato (Abad et al., 1997). In order to test hypothesis, it was first necessary to engineer plants with a trangenic bO gene, then to test whether or not this gene brought about the same cellular effects that occur in plants, which usually have the bO gene. Leaf tissue samples were analyzed for cell death by staining them with trypan blue and observing them under a microscope a method borrowed from Hebers et al., 1996. From these observations the complexity and irregularity of the lesion-mimic phenotype was deduced.
The next step in the analysis investigated whether the lesion-mimic phenotype in potato was somehow linked to the induction of pathogenesis-related genes. PR genes were found dramatically over-expressed in lesioned tissue displaying high levels of bO. Levels of free and total salicylic acid, also, increased tremendously compared to levels in the control plants. Finally, the genetically engineered plants had to be tested for resistance to pathogens. Abad et al, tested the engineered lesion-mimic phenotype for resistance to pathogens which the regular wild-type phenotype would be susceptible to.
Results suggested that the ability to transfer immunity against a pathogen from plant to plant was extremely limited. The costs of introducing the bO gene into plants, in terms of growth and development, discounted the observed benefit. Could the lesion-mimic mechanism, by which plants resist pathogens, be manipulated such that it could be used to produce genetically modified plants that would improve plant crops? Current research suggests that, no, the bO gene and the bO-induced lesion-mimic phenotype would not be beneficial if employed in agriculture today. Cell death induced by the bO gene does afford plant species, including but not limited to tobacco, resistance to the US1 isolate of P. infestans.
Plants, however, are not afforded resistance to the more potent isolate of P. infestans, namely US8. In fact, plants are also still susceptible to potato virus X and Erwinia carotovora, despite the presence of the bO gene. The bO gene is limited in its ability to confer resistance to pathogens. This is not reason enough to discredit the bO gene and lesion-mimic phenotype as possible solutions to limit infection of plants by pathogens. The deleterious effects on the growth of transgenic plants with the bO gene, however, discount any possible utility the introduction of the bO gene into at risk plants would have in an agricultural setting.
Beyond a utilitarian analysis of practical costs and benefits, it could also be argued that public distaste for genetically modified foods is sufficient reason, in itself, to limit the widespread implementation of this type of technology in an agricultural setting. Gradual implementation of only the best techniques will not only bring about a better public support for such technologies, but will also allow us to better gauge the effects of genetically modified foods.