Engineering Plants to Influence Rhizosphere Biochemistry: Potential Benefits to Plant Nutrition.
Timothy S. George, CSIRO Plant Industry, GPO Box 1600,, Canberra, ACT 2611, Australia
A large proportion of endogenous soil Phosphorus (P) occurs in organic forms, of which derivatives of inositol phosphates constitute a major fraction. The bioavailability of inositol phosphates depends on their mineralization by extracellular phytases. However, we currently have a poor understanding of the interactions between phytases and inositol phosphates in the rhizosphere. Transgenic plants which exude phytase to the rhizosphere allow us to directly manipulate rhizosphere biochemistry and have potential to elucidate mechanisms of organic P turnover inconceivable with other less strictly controlled experimental material. With phytate as the sole P source in agar, transgenic T. subterraneum exhibited significantly improved growth and P accumulation (2.9-fold increased shoot P content) compared to controls. This was attributed to a 77-fold increase in phytase activity exuded to the rhizosphere. However, when grown in soil these unique characteristics were compromised. In that, transgenic T. subterraneum had only 1.2-fold greater P accumulation than controls. In contrast, transgenic N. tabacum accumulated up to 1.5-fold more P than controls, but only in soils amended with inositol phosphate. In unamended soils all plants depleted phytate (phytase-labile P), which was determined by its lability to a non-specific phytase (Sigma-Aldrich). Over the growth period 60% of phytase-labile P was depleted from a low P-sorption rhizosphere soil, while only 15% disappeared from moderate P-sorption rhizosphere soils. Transgenic plants were more effective, depleting 80% of phytase-labile P, but only in the low P-sorption soils. This suggests that the availability of inositol phosphates in the rhizosphere is key to improved P nutrition of phytase exuding plants. Another potential explanation for limited responses of transgenic plants in soils is that phytase activity is compromised in the rhizosphere environment. Phytase collected from the roots of plants expressing a phytase gene (phyA) from Aspergillus niger disappeared from soil solution within 10 minutes of addition to an unamended soil. Between 58 and 88% of the activity lost from soil solution, upon addition to bulk soils, was immediately recoverable on the soil solid phase. Moreover, up to 48% of added phytase was still accountable after 28 days. However, when added to soils taken from the rhizosphere of transgenic plants, phytase activity remained in solution. Despite the apparent protection by adsorption, immobilisation to the solid phase will presumably limit phytase interactions with inositol phosphates. Importantly solubility of phytase appears to be controlled by pH, such that phytase added with increasingly alkaline pH was increasingly recovered in solution. Partitioning of enzyme activity to the solution phase with increased pH is attributed to enthalpic forces causing greater electrostatic repulsion above the isoelectric point (pI) of the protein. Significantly, when adsorption characteristics of two phytases with different pI were compared in acidic soils, major distinctions were observed. Phytase with a relatively low pI (pH 3.6 from Peniophora lycii) remained in solution in a range of soils (pH 4.5 to 5.0), whereas phytase with a higher pI (pH 4.8; from Aspergillus niger) was totally adsorbed. Moreover, we have found that the phytase with low pI is approximately twice as effective in hydrolysing inositol phosphates in soil suspensions and much more effective at hydrolysing endogenous soil phytate. However, this soluble phytase was much more rapidly degraded, further emphasising the protection afforded to enzymes by adsorption to the solid phase in the rhizosphere. The biochemical characteristics of phytases produced by different organisms have obvious implications for their behaviour in soil environments and thus their efficacy for mineralization of inositol phosphates. Maintaining phytases in solution apparently enhance their ability to interact with immobilized substrates. Future work will concentrate on expressing genes in plants for the production of phytases with biochemical traits that effect their interaction with soil, such as low pI. This will allow better understanding of the factors that contribute to the biological availability of inositol phosphates in soil and improve the ability of plants to acquire phosphorus by overcoming limitations imposed by the rhizosphere environment.