Salinity tolerance of barley expressing an Arabidopsis vacuolar pyrophosphatase gene

Rhiannon K. Schilling^1,2, Petra Marschner², Yuri Shavrukov^1,2, Bettina Berger^2,3, Mark Tester^1,2,3, Stuart J. Roy^1,2 and Darren C. Plett^1,2

¹ Australian Centre for Plant Functional Genomics, ² School of Agriculture, Food and Wine, The University of Adelaide, ³ The Plant Accelerator, Australian Plant Phenomics Facility, The University of Adelaide

GRDC Project code: GRS10044

Keywords: barley, AVP1, grain yield, salinity, GM field trials

Take home messages

• Saline soils reduce the grain yields of cereal crops
• Preliminary field trial results suggest barley over-expressing AVP1 has greater shoot biomass and grain yield in a saline field than barley plants without AVP1
• Further saline field trial evaluation of transgenic AVP1 barley lines is ongoing

Background

Saline farmland containing high amounts of sodium chloride (NaCl) can reduce the productivity of cereal crops. Salt stress restricts water uptake and increases leaf senescence causing stunted growth and an overall reduction in tiller number and grain yield (Munns and Tester, 2008). The development of cereal crop varieties with improved salinity tolerance is needed to help increase crop productivity in saline soils.

The over-expression of an Arabidopsis vacuolar pyrophosphatase gene (AVP1) has previously been shown to improve the salinity tolerance of Arabidopsis, alfalfa, bentgrass, cotton and rice (Bao et al., 2009; Gaxiola et al., 2001; Li et al., 2010; Pasapula et al., 2011; Zhao et al., 2006). This improved salt tolerance of transgenic plants over-expressing AVP1 was attributed to an enhanced electrochemical gradient across the vacuolar membrane allowing greater sequestration of sodium (Na⁺) into cell vacuoles. The sequestration of Na⁺ into a cell vacuole can reduce Na⁺ toxicity and facilitate water uptake helping to improve plant growth.

The aim of this project was to evaluate the shoot growth and grain yield of barley over-expressing AVP1 in a saline field to test whether or not this gene is able to improve the salinity tolerance of this cereal crop.

Saline Field Trial Evaluation of Transgenic AVP1 Barley

A small scale field trial of transgenic barley (cv. Golden Promise) with constitutive over-expression of AVP1 was conducted at a saline field site near Kunjin, Western Australia. An EM38 map of the field site and electrical conductivity (EC_1:5) measurements of soil cores (0-10 cm) were used to identify field areas of low (EC_1:5 = 161 ± 11 μS/cm) and high (EC_1:5 = 1231 ± 155 μS/cm) salinity. The field trial design was completely randomised with two plots (1.2 m width × 2 m length) per line in two salt treatments (low and high salt). Plots were sown in July 2011 with standard agronomic practices used. For each plot, the shoot biomass and the number of tillers of six randomly selected plants were measured. The youngest emerged leaf blade was also collected for solute measurements. Prior to harvest, grain was sampled from six randomly selected plants from each plot with the number of grain heads, number of individual grains and grain weight per plant recorded.

Preliminary results indicate that the barley over-expressing AVP1 has a larger shoot biomass compared to wild-type plants in the low and high salt field areas (Figure 1). The grain yield per plant was 5 to 34 % higher in the transgenic AVP1 barley lines than wild-type plants in the low salt area. While in the high salt area, a combination of high salt and water-logging stresses greatly reduced the growth of all plants. Promisingly, in these high salt and water-logging conditions, the barley lines over-expressing AVP1 were able to maintain better shoot growth (Figure 1) and higher grain yield per plant compared to the wild-type barley. The number of heads and individual grains of barley plants over-expressing AVP1 in the high salt field was 16 to 58 % and 76 to 85 % higher than wild-type, respectively.

Figure 1. Saline field trial plots of wild-type (cv. Golden Promise) and T₄35S:AVP1 barley. All plots were randomly located in a low salt (EC_1:5 = 161 ± 11 μS/cm) and high salt (EC_1:5 = 1231 ± 155 μS/cm) field area with two replicate plots, for comparative purposes the plot images in this figure have been grouped by genotype.

Summary and Future Work

This project established a small scale saline field trial to test transgenic barley plants. Preliminary results suggest that the over-expression of AVP1 improves the growth of barley leading to an increase in shoot biomass and grain yield in a saline field. Future field trials testing the transgenic AVP1 barley lines over multiple seasons across different saline field sites and with greater plot replication is required. Additionally, a commercially relevant cultivar expressing this gene is also needed. Ultimately, this research is helping to develop a salt tolerant barley crop variety with improved grain yield for Australian farmers in the future.

Acknowledgements

The authors wish to acknowledge and thank Kalyx Australia (Perth), Precision Agronomics Australia (Esperance), Jan Nield and Andrew Jacobs (ACPFG) for their assistance in establishing and conducting the GM field trial. RS also acknowledges a GRDC Grains Industry Research Scholarship.

References

Bao, A.K., Wang, S.M., Wu, G.Q., Xi, J.J., Zhang, J.L. and Wang, C.M. (2009) Overexpression of the Arabidopsis H⁺-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Science 176, 232-240.

Gaxiola, R.A., Li, J.S., Undurraga, S., Dang, L.M., Allen, G.J., Alper, S.L. and Fink, G.R. (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H⁺-pump. Proceedings of the National Academy of Sciences of the United States of America 98, 11444-11449.

Li, Z.G., Baldwin, C.M., Hu, Q., Liu, H. and Luo, H. (2010) Heterologous expression of Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.). Plant Cell and Environment 33, 272-289.

Munns, R. and Tester, M. (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651-681.

Pasapula, V., Shen, G., Kuppu, S., Paez-Valencia, J., Mendoza, M., Hou, P., Chen, J., Qiu, X., Zhu, L., Zhang, X., Auld, D., Blumwald, E., Zhang, H., Gaxiola, R. and Payton, P. (2011) Expression of an Arabidopsis vacuolar H⁺-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions. Plant Biotechnology Journal 9, 88-99.

Zhao, F.Y., Zhang, X.J., Li, P.H., Zhao, Y.X. and Zhang, H. (2006) Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1. Molecular Breeding 17, 341-353.

Contact details
Rhiannon Schilling
ACPFG, PMB 1 Glen Osmond, SA 5064

(08) 8313 7161
rhiannon.schilling@acpfg.com.au