Department of Biochemistry
Assimilation of fixed N, expression and role of arginine breakdown enzymes, Putilization and non-conventional means of improving soybean performance.
Joe Polacco's group takes a molecular genetics and functional genomics approach to the study of nutrient sensing, assimilation and movement in plants (soybean and Arabidopsis). The focus is on nitrogen and phosphorous, the two nutrients most limiting to plant growth. A third 'nutrient' is nickel, whose only known function is to form the active metallocenter of urease. A study of Ni uptake and movement is related to phytoremediation- the use of plants to cleanse soils of toxic contaminants, usually heavy metals. Natural hyperaccumulators of Ni provide examples of how this metal may move in soybean.
A series of urease-negative mutants in soybean has been exploited to determine the origins of urea and the developmental and physiological conditions influencing its accumulation. Urea, which is 47% nitrogen, is not a waste product in plants; metabolically derived urea must be recycled by urease action. The urease-negative mutants have revealed that urea is derived both from arginine and from purines. Purine degradation is extremely important to soybean performance since the soybean nodule is a purine biosynthetic factory, eventually oxidizing purines to ureides. In soybean, most fixed nitrogen is 'presented' to green tissues as ureides, and the route(s) of ureide conversion to utilizable ammonia have long been debated, and models have been proposed which related the nature of the degradative pathway to sensitivity of N-fixation to water stress.
Polacco's group has demonstrated that the soybean has only a single ureide degradative pathway, and is examining other factors that can influence drought-resistance in the nodule.
Polacco's group has demonstrated that the soybean embryo temporally separates arginine production from its breakdown, thus avoiding a futile arginine/urea cycle. Part of this separation involves controlling the movement of arginine into the mitochondrion, the site of arginase. His lab has isolated and characterized two mitochondrial transporters of arginine and other basic amino acids.
A current emphasis is to examine possible links between controls on arginine pools (via arginases and mitochondrdial arginine transporters) and the generation of nitric oxide (NO). It is becoming increasingly apparent that NO is involved in a wide array of plant developmental and defense pathways.
Ni metabolisms is being examined, starting from the role of Ni and various accessory proteins in activating apo-urease and extending to genetic identification of factors controlling Ni uptake and movement. It is of interest that one of the soybean accessory proteins resembles a protein required for Ni activation of hydrogenase, a bacterial enzyme not yet identified in plants.
Assimilation of Fixed Nitrogen (N)
The N2 that is fixed in the root nodule is presented to the plant as ureides. These compounds are produced from purines, normally employed by plants as components of DNA, RNA, energy transfer compounds (GTP, ATP) and co-factos. We have defined some of the last steps in the conversion of ureides into ammonia, the precursor of nitrogenous compounds in the green aerial portions of the plant. Currently, there is a hypothesis that there are alternative routes of breakdown of the ureide allantoate: "Our" pathway invokes direct conversion of half of the allantoate N to ammonia. A second pathway, operative in plants which can fix N2 under water-deficit, converts allantoate to urea, which is subsequently converted to ammonia. The key feature of this hypothesis is that Manganese (Mn) is required for the allantoate to ammonia conversion while it is not required for the allantoate to urea conversion. Since Mn moves less freely under water-deficit, allantoate accumulates in the ammonia-producing varieties in which it feedback inhibits fixation. We are currently testing this model. The goal is breeding and biotech targets for making soybean more drought tolerant.
Expression and Role of Arginine Breakdown Enzymes
Arginine (Arg) is the major nitrogenous compound in soy protein. In addition, it is a precursor to such imporant metabolites as polyamines and nitric oxide (NO). A harmful fate of Arg is its premature conversion to urea and ornithine by the arginase reaction. If this reaction occurred in the developing soybean seed, it would represent a tremendous losses of valuable N and protein content. One means of control of the fate of Arg is its movement to various subcellular compartments. For example, if Arg had unimpeded movement into the mitochondrion, a common cellular energy generator, it would be readily converted to ornithine and urea by the mitochondrial arginase activity. This has led us to study the "portals" for Arg in the mitochondrial membrane. We have identified two Arg transporters and are trying to control their expression in soybean so that more Arg accumulates in the leaves and eventually makes its way to the mature seed. We are also trying to achieve the same goal by controlling levels of cellular arginase activity. A component of these studies is an examination of how the enzyme urease is activated by Ni.
Of the six mineral macronutrients, phosphorus (P) is the second limiting, after N, in soybean productivity, this in spite of many soils having considerable P content. The problem is that soil P is in both mineral and organic forms that are unavailable to the plant. Since free phosphate diffusion in the soil is also low, the rhizosphere tends to be extremely depleted in soluble P. A conundrum is that in order to avoid crop losses due to P-limitation, farmers are obliged to rely on P fertilization of P-containing soils which is expensive, harmful to the environment and challenged by diminishing ag lime reserves. To improve soybean P nutrition our goal is to understand how soybean senses, takes up and assimilates P. To this end we are taking a genomics approach in which we use microarray analysis to observe how each of 45,000 soybean genes responds to P-deficiency. We are manipulating not only P availability, but also potential regulatory genes (i.e. mutants). The goal is to identify key genes whose increased expression can make soybean less dependent on P fertilization, perhaps even more efficient in extracting normally unavailable P from the soil.
Unconventional Means of Improving Soybean Performance
We have identified a commensal, leaf surface-dwelling bacterium that can stimulate germination of aged soybean seeds. This bacterium is pink (because of carotenoids) and can utilize one-carbon compounds (e.g. methanol, methylamine, formic acid) as sole energy and carbon sources, and its various isolates fall in the genus Methylobacterium. There is also strong circumstantial evidence that foliar application of these bacteria on field soybeans, esp in dry years, increases yield. Yield stimulation is possibly due to the secretion of cytokinins by these bacteria. Cytokinins are a class of senescence reverting hormones, and we have determined that a powerful cytokinin, trans-zeatin, is secreted by the bacteria. We are seeking to further our understanding of bacterial-plant communication, and how we may manipulate to improve soybean performance.