Transgenic expression of phytase and acid phosphatase genes in alfalfa (Medicagosativa) leads to improved phosphate uptake in natural soils

Abstract

Alfalfa (Medicagosativa L.) is one of the most widely grown crops in the USA. Phosphate (P) deficiency is common in areas where forage crops are grown. To improve the use of organic phosphate by alfalfa, two Medicagotruncatula genes, phytase (MtPHY1) and purple acid phosphatase (MtPAP1), were overexpressed in alfalfa under the control of the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Root enzyme activity analyses revealed that although both genes lead to similar levels of acid phosphatase activities, overexpression of the MtPHY1 gene usually results in a higher level of phytase activity than overexpression of the MtPAP1 gene. The MtPT1 promoter was more effective than the CaMV35S promoter in regulating gene expression and extracellular secretion under P-deficient conditions. Measurement of growth performance of the transgenic lines further proved that the best promoter-gene combination is the MtPHY1 gene driven by the MtPT1 promoter. Compared to the control, the plants with high levels of transgene expression showed improved growth. The biomass of several transgenic lines was three times that of the control when plants were grown in sand supplied with phytate as the sole P source. When the plants were grown in natural soils without additional P supplement, the best performing transgenic lines produced double the amount of biomass after 12 weeks (two cuts) of growth. Transgene effects were more obvious in soil with lower pH and lower natural P reserves than in soil with neutral pH and relatively higher P storage. The total P concentration in leaf tissues of the high-expressing transgenic lines was significantly higher than that of the control. The transgenes have great potential for improving plant P acquisition and biomass yield in P-deficient agricultural soils. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9628-0) contains supplementary material, which is available to authorized users.

Fig. 1

APase activity of transgenic and control (CTL) alfalfa plants. The first letter of each plant name represents the gene construct used for producing the transgenic line. a Activities of whole root extracts of the MtPHY1 plants. b Activities of whole root extracts of the MtPAP1 plants. c Activities of root saps of the MtPHY1 plants. d Activities of root saps of the MtPAP1 plants. e Activity correlation between whole root extracts and root saps of the MtPHY1 plants. f Activity correlation between whole root extracts and root saps of the MtPAP1 plants. Data are presented as means ± SE (n = 6)

Fig. 2

Phytase activity of transgenic and control (CTL) alfalfa plants. The first letter of each plant name represents the gene construct used for producing the transgenic line. a Activities of whole root extracts of the MtPHY1 plants. b Activities of whole root extracts of the MtPAP1 plants. c Activities of root saps of the MtPHY1 plants. d Activities of root saps of the MtPAP1 plants. e Activity correlation between whole root extracts and root saps of the MtPHY1 plants. f Activity correlation between whole root extracts and root saps of the MtPAP1 plants. Data are presented as means ± SE (n = 6)

Fig. 3

Biomass of transgenic alfalfa and control plants in sand supplied with phytate as the only P source. Data are presented as the means ± SE (n = 4) of the dry weights

Fig. 4

Biomass and total P concentration in leaf tissues of transgenic alfalfa and control plants in two types of soils without any P supply. a Soil 1 has a low pH and low P reserves. b Soil 2 has a neutral pH with higher P reserves than soil 1. For both soils, the first cut was done 8 weeks after planting and the second cut was done 4 weeks after the first cut. Data are presented as means ± SE (n = 6) of the dry weight. c Total P concentration in leaf tissues obtained from second cut of transgenic alfalfa and control plants grown in soil 1 and soil 2. Data are presented as means ± SE (n = 4)