Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate.

Lia R Valeeva,Eugene V Shakirov,Margarita R Sharipova,Chuluuntsetseg Nyamsuren

doi:10.3389/fpls.2018.00186

Lia R Valeeva, Eugene V Shakirov + Show 2 more

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https://doi.org/10.3389/fpls.2018.00186

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Abstract

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

Highlights

Phosphorus is an essential element required for plant growth and development
We demonstrate that transgenic plants with high levels of 168phyA and PaPhyC phytase activity were able to efficiently utilize phytate from perlite and hydroponic media resulting in larger rosette diameter and leaf area, and higher phosphorus content than in control plants grown in the same conditions
To test the effects of histidine acid phosphatases (HAPs) and BPP phytase expression in A. thaliana on plant morphology and physiology, the coding regions of phytase genes paPhyC from Pantoea agglomerans and 168phyA from Bacillus subtilis were codon-optimized for expression in plants (Chuluuntsetseg et al, 2015; Valeeva et al, 2015), fused with the carrot extensin leader sequence (Supplementary Figures S1, S2) and placed under the control of the full 2 kb inducible root-specific Pht1;2 promoter from A. thaliana (Mudge et al, 2002, 2003)

Summary

Introduction

Phosphorus is an essential element required for plant growth and development. Most plants acquire phosphorus from soil in the form of inorganic phosphate, but soluble phosphate reserves in many agricultural soils rapidly decline due to highly demanding agricultural practices. Crop production relies heavily on application of rock phosphate fertilizer to improve soil phosphorus availability. This approach is not sustainable on the long-term, and several recent analyses forecast that rock phosphate deposits worldwide will be largely depleted by the end of this century (Van Vuuren et al, 2010; Gaxiola et al, 2011). While plants do possess various types of phytases, they are largely intracellular or their expression is often restricted to a specific phase of plant development (Hegeman and Grabau, 2001; Richardson et al, 2001; Konietzny and Greiner, 2002; Greiner, 2007) and cannot substantially contribute to improved plant growth in conditions of limited inorganic phosphate availability in soil

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