Evaluation of the effects of AtCIPK16 expression on the salt tolerance of barley and wheat

Abstract

Soil salinity is a major constraint to crop production in Australia. This has prompted the need to produce salt tolerant cereal cultivars, through the understanding of genes involved in salt tolerance mechanisms and manipulating their expression levels. Arabidopsis thaliana Calcineurin B-like Interacting Protein Kinase 16 (AtCIPK16) has been identified as a gene involved in sodium (Na+) exclusion. Analysis of AtCIPK16 alleles from Arabidopsis ecotypes suggests variances in expression are due to differences in the promoters. Experiments in Arabidopsis, barley and wheat (preliminary) have illustrated that AtCIPK16 overexpression can enhance biomass production through increased Na+ exclusion, although its full effect in barley and wheat has yet to be properly characterised in both greenhouse and field environments. The first focus of this project evaluated the salt tolerance of 35S:AtCIPK16 barley (cv. Golden Promise) grown under low and high salinity field conditions in 2013 and 2014 at Kunjin, Western Australia. Comparisons between years were difficult due to waterlogging of the 2013 high salt site and the increased variability in plot establishment in 2014. 35S:AtCIPK16 barley lines had varying responses to high salt conditions depending on the annual rainfall. Results showed Na⁺ and Cl⁻ exclusion in certain lines, although this correlated with decreased biomass and yield in high rainfall years. AtCIPK16 expression also increased Na⁺ and Cl⁻ exclusion in 2012 (a low rainfall year) which instead lead to increasing plant growth and yield. The second focus of this project aimed to fully characterised the effects of the constitutive expression of Ubi:AtCIPK16 in wheat (cv. Gladius). Despite conducting three hydroponic experiments, no definitive conclusions about the effects of AtCIPK16 expression on wheat salt tolerance could be drawn. Although, one sibling transgenic line showed increased Na⁺ and Cl⁻ exclusion from both root and shoot tissue accompanied by larger biomass under 200 mM salt stress. Despite this finding several factors hinder the analysis of data including the high number of null segregants, considerable variability between siblings of the same transformation event and minimal transgene expression. The third focus of this project aimed to investigate expression differences between two AtCIPK16 alleles from the Arabidopsis ecotypes Bay-0 and Shahdara. Since the only differences between the two alleles was a 10 base pair deletion in the Bay-0 promoter, it was hypothesised this deletion was the reason for the increased expression of AtCIPK16 in Bay-0 as it forms a TATA box (TATATAA). The aim of this project was to alter the expression of each allele by: mutating the last A to a T, removing the TATA box in Bay-0, and mutating the T after the TATATA sequence to an A in Shahdara, forming a TATA box without the deletion. Through PCR mutagenesis the required point mutations were introduced into portions of the two promoter alleles, however due to technical difficulties and time constraints the point mutations were not introduced back into the full promoter constructs driving GFP. It was therefore unable to be determined if the point mutations to the TATA box would indeed affect AtCIPK16 expression.