Root cell-type specific expression of multiple salinity tolerance genes to alter plant shoot sodium accumulation

Abstract

Increasing soil salinity of agricultural land is of growing concern world-wide as excessive soil salinity has a detrimental effect on growth and yield of many plant species of agricultural importance. The accumulation of sodium ions (Na⁺) from saline soils into the shoots of crop plants contributes to the negative effect salinity has on plant growth in cereals. In recent years, many molecular targets involved in Na⁺ transport in plants have been identified in a number of species. Genetic modification (GM) utilising these genes may enable manipulation of Na⁺ transport with an aim of reducing Na⁺ accumulation in the shoot. Constitutive and/or tissue-specific over-expression (OX) of such genes in transgenic plants can prove beneficial in reducing Na⁺ shoot accumulation and improve plant salinity tolerance in some cases. However, further reductions could be made by fine tuning Na⁺ transport through the plant by co-expressing multiple salinity tolerance associated genes of interest (GOI) in specific root-cell types. To date, this has proved difficult. Previously generated barley (Hordeum vulgare c.v Golden Promise) lines with putative cell-type specific OX of salinity tolerance associated GOIs, High Affinity K⁺-Transporter 1;5 (HvHKT1;5) and vacuolar H⁺-pyrophosphatase 1 (HvHVP1), were screened in saline hydroponics to assess for improvements in salinity tolerance. Lines with the simultaneous root-cell-type specific OX of both HvHKT1;5 and HvHVP1 were developed through hybridisation and assessed for improved salinity tolerance. Although no significant improvements were identified in both the single- or dual-GOI transgenic lines, this approach could be used for other transgenic lines with cell-type specific OX of other GOIs combinations. The role of vacuolar H⁺-pyrophosphatase 1 (AtAVP1) was re-examined when over-expressed in the root-epidermal and –cortical cell types in the model plant species Arabidopsis thaliana. OX of AtAVP1 in these cell-types was thought to improve Na⁺ sequestration and there-by improve salinity tolerance. However, saline hydroponics assays of lines with root-epidermal and/or –cortical OX of AtAVP1 failed to identify improvements in plant salt tolerance or Na⁺ uptake, suggesting that AtAVP1 contributes little to Na⁺ sequestration in these cell-types. Finally, a system that would allow the cell-type specific over-expression of different GOIs in different root cell-types was developed. Such a system would allow the trialling different gene combinations to identify combinations that would allow more targeted manipulation of Na⁺ transport throughout a plant and alter salinity tolerance. This work was carried out in the model plant species, Arabidopsis thaliana, and cell-type expression was enabled through the use of dual GAL4 and HAP1 enhancer-trap systems and trans-activation constructs. Lines and constructs were developed to allow the cell-type specific OX of selected GOIs, however testing of dual salinity tolerance GOI lines was not achievable during the timeframe of this project.