Nitrilase 2: insight into its regulation in Arabidopsis and its potential for improving maize salt tolerance

Foreman, Nina-Courtney

Nitrilase 2: insight into its regulation in Arabidopsis and its potential for improving maize salt tolerance

Thesis / Dissertation

2023

Abstract

By the year 2050, it is estimated that more than 50% of the arable land worldwide will be too saline to sustain the growth and productivity of many crop plants. Soil salinisation threatens food security because it reduces crop yield and quality. Therefore, to increase food production for the growing population, we need to improve crop salt tolerance. To do this, we need a better understanding of inherent plant molecular responses to salt stress in order to engineer crops with enhanced salt tolerance. Previously, our group has shown that in Arabidopsis, salt-specific genes are enriched in the gene ontology term “response to auxin stimulus”, and auxin levels increase under saline conditions. Nitrilase 2 was identified as the biosynthetic gene possibly responsible for these changes in auxin accumulation as AtNit2 expression was elevated specifically under saline conditions. Additionally, AtNit2 overexpression lines were more salt tolerant. As AtNit2 is a candidate for enhancing plant growth under saline conditions to improve salt tolerance, it is important to understand how this gene is regulated and this was the main aim of this research project. The AtNit2 promoter region was analysed and five MYELOBLASTOSIS (MYB) transcription factor (TF) binding sites were identified. Interestingly, two MYB TFs were upregulated specifically in response to salt in our experiments. These two TFs, AtMYB2 and AtMYB30 were functionally characterised in Arabidopsis to investigate whether they might be upstream of AtNit2 in the plant salt stress response pathway. Overexpression of AtMYB2 in Arabidopsis did not lead to altered AtNit2 expression or biomass production under saline conditions, nor was binding of AtMYB2 to the AtNit2 promoter observed in a yeast one-hybrid (Y1H) assay, suggesting that it may not be involved in AtNit2 regulation. Although AtMYB30 did not bind directly to the AtNit2 promoter in the Y1H assay, AtMYB30 overexpressing plants were more salt tolerant and showed increased expression of AtNit2 under control and saline conditions. An atmyb30 T-DNA mutant line also showed a reduction in salt tolerance, however AtNit2 was still upregulated under saline conditions in the atmyb30 T-DNA mutant lines. Overall, this data indicates that AtMYB30 might play an indirect role in AtNit2 regulation. To identify TFs that can bind to the AtNit2 promoter, a Y1H TF library screen approach was used. Six TFs were identified: HOMEOBOX PROTEIN 34 (AtHB34), HOMEOBOX PROTEIN 24 (AtHB24), HOMEOBOX PROTEIN 28 (AtHB28), HIGH MOBILITY GROUP BOX PROTEIN 9 (AtHMGB9), GLABRA 2 (AtGL2), and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7 (AtSPL7). Two of these TFs were further characterised: AtHMGB9 and AtSPL7. Reporter assays in Arabidopsis mesophyll protoplasts showed that AtHMGB9 was able to bind to and negatively regulate AtNit2 promoter activity in planta. However, athmgb9 mutant lines displayed only slightly increased AtNit2 expression under saline conditions. While transfection of protoplasts with AtSPL7 did not lead to changes in AtNit2 promoter:reporter activity, atspl7 lines showed slightly increased AtNit2 expression indicating that AtSPL7 may play a role in negatively regulating AtNit2 expression but may require other co-factors to do so in planta. To determine whether Nit2 regulation is also important for maize salt tolerance, preliminary analysis of the maize Nit2 homolog, ZmNit2, showed that ZmNit2 expression was induced in response to salt in both root and shoot tissue in a dose-dependent manner, implying that auxin might play a role in salt tolerance across different plant species. Overexpressing ZmNit2 was sufficient to increase salt tolerance of two-week old Arabidopsis plants, indicating that ZmNit2 may play a role in the maize response to salt stress early in development and therefore suggests that genes identified in Arabidopsis may be appropriate targets for manipulation in crop plants, such as maize. Overall, this study provides novel insights into the regulation of AtNit2 by identifying several TFs that may bind to and regulate AtNit2 expression. It also shows that ZmNit2 is able to improve Arabidopsis salt tolerance and indicates a potential role in improving maize salt tolerance.

Keywords

Molecular and Cell Biology

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PhD / Doctoral

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