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

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.

A root‐associated purple acid phosphatase, SgPAP23, mediates extracellular phytate‐P utilization in Stylosanthes guianensis

“…In attempts to enhance extracellular phytate‐P utilization in plants, great success has been realized through overexpression of a secreted signal peptide fused with a phytase or PAPhy gene (either from microorganisms or plants), with examples including overexpression of ex:AtPAP15 in soybean, ex:PHY in potato ( Solanum tuberosum ), ex:phyA and ex:appA in Brassica napus , and ex:phyA , ex:PHY‐US417 , and ex:PaPhyC in Arabidopsis (Belgaroui, Berthomieu, Rouached, & Hanin, ; Richardson, Hadobas, & Hayes, ; Valeeva, Nyamsuren, Sharipova, & Shakirov, ; Wang et al, ; Wang, Ye, Ding, & Xu, ; Zimmermann et al, ). On the other hand, few studies have been conducted to investigate natural variation in RA‐Phy activity and the capability to utilize phytate‐P within a plant species, which are necessary steps toward developing cultivars that are highly efficient in extracellular phytate‐P utilization.…”

Phosphobacteria as key actors to overcome phosphorus deficiency in plants

“…An additional approach to achieve P mineralisation that goes beyond the use of phosphobacteria and purified microbial phytases is based on genetic transformation of plants with phytase genes of microbial origin. Studies in this field involved the transformation of a variety of plant species with fungal and bacterial phytases of the HAP type (Valeeva et al, 2018).…”

“…Tobacco and A. thaliana transformation with B. subtilis BPP led to increased plant growth on both species and increased P content in A. thaliana (Belgaroui et al, 2016; Lung et al, 2005; Yip et al, 2003). A recent study that compared the advantages of transforming A. thaliana with HAP and BPP concluded that, in principle, both approaches render similar results but their efficacy can be differentially affected by growth conditions (Valeeva et al, 2018). The above‐mentioned studies suggest that the expression of bacterial phytases in transgenic plants could provide an important means for improving growth of cultivated crops under conditions of P deficiency.…”

Native and non-native host assessment towards metabolic pathway reconstructions of plant natural products

“…Multiple heterologous expression systems have been developed from simple prokaryotes such as E. coli [ 73 ] to higher eukaryotes, including plant [ 74 ] and mammalian cells [ 75 ]. Among the various eukaryotic heterologous hosts, single plant cells appear to be more promising with regard to error-free PTMs for the synthesis of secondary plant bioactives ( Table 1 ).…”