Browsing by Author "Ingle Robert"

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    Open Access
    Investigating the molecular mechanism whereby auxin modulates Arabidopsis thaliana growth under salinity stress conditions
    (2023) Ferrandi, Paul; Donaldson, Lara; Ingle Robert
    Soil salinization is detrimental to plant growth and yield, and an increasing global problem in irrigated agricultural land. Salinity imposes both an osmotic and ionic stress on plants, the latter of which is unique to salt stress. Previous work has indicated that the plant growth hormone auxin could play an important role in ionic stress tolerance: transcriptome profiling revealed that the genes differentially expressed in response to ionic stress were enriched with the gene ontology term ‘Response to auxin'. Additionally, expression of the auxin biosynthesis gene, Nitrilase 2 (NIT2), was also specifically up-regulated in response to ionic stress. A NIT2 overexpressing line (35S::NIT2) has been shown to display improved germination and growth, as well as increased auxin levels compared to wild-type plants under saline conditions. The hypothesis thus is that salt stress induces NIT2 expression, thereby increasing auxin biosynthesis, which results in the activation of the plasma membrane (PM) H+-ATPase pump and subsequent activation of expansion growth mediated by expansins. Additionally, the pump activation could enhance the proton motive force for ion transport across the PM, thereby affecting ion homeostasis during salt stress. The aim of this study was to investigate this proposed molecular mechanism whereby auxin modulates plant growth in response to salt stress. Measurement of Na+ and K+ content revealed that 35S::NIT2 plants had improved root and shoot Na+/K+ ratios compared with wild-type (No-0 ecotype) plants, under saline conditions. This altered ion homeostasis might be a consequence of differences in the expression of several Na+ and K+ transporter/channel genes between the two lines. Of particular interest, under saline conditions, the 35S::NIT2 line had increased root HKT1;1, AKT1, and SKOR expression, as well as decreased GLR2.3 expression; and increased shoot NHX3, SOS1, and KUP1 expression. Together these results provide evidence that improved Na+/K+ homeostasis underlies the increased salinity tolerance of the 35S::NIT2 line. Proteomic analysis of enriched PM protein fractions isolated from the shoots of salt-stressed and untreated 35S::NIT2 and No-0 plants revealed genotype-specific differences in salt-responsive PM protein abundance, as well as differences between the two lines under both conditions. Gene ontology analysis revealed that several of the identified salt- responsive proteins were assigned with GO terms related to auxin and the ‘response to salt stress'. This included NIT2, which was up-regulated in response to salt in both 35S::NIT2 and No-0 plants, supporting the hypothesis that auxin biosynthesis is increased via NIT2 to modulate growth in saline conditions. Notably, far fewer salt-responsive proteins were identified in the 35S::NIT2 line than in No-0. A subset of the salt-responsive proteins identified in No-0 displayed higher constitutive expression in the 35S::NIT2 line under control conditions, and were not differentially expressed in response to salt (ERD7, PATL1, ABCC4, DRP2B). I propose that the processes that these proteins regulate, or are involved in, could be constitutively active in the 35S::NIT2 line, and that they could contribute to the increased salt- tolerance of the 35S::NIT2 line. This is the first report of salt-responsive changes in the PM proteome of Arabidopsis shoots, as well as the differences in the PM proteome of the 35S::NIT2 line that could contribute towards its improved salinity tolerance. Finally, Expansin 11 (EXPA11) was identified as a candidate growth effector acting downstream of auxin-induced PM H+-ATPase activation, as its expression was shown to be significantly higher in the 35S::NIT2 line than in No-0, and was further up-regulated in response to salt stress. Attempts to knock-down EXPA11 expression in the 35S::NIT2 line proved unsuccessful, with the transgenic 35S::EXPA11amiRNA lines not displaying any reduction in EXPA11 expression, or an altered phenotype. Transgenic lines overexpressing EXPA11 (35S::EXPA11) displayed increased hypocotyl elongation, improved seedling growth in untreated, salt, and osmotic stress conditions, and increased shoot fresh weight in older plants under saline conditions. Furthermore, the 35S::EXPA11 plants displayed a greater leaf surface area, with increased epidermal and palisade mesophyll cell areas. Collectively, these results indicate that EXPA11 plays a role in promoting growth in multiple tissues across different developmental stages, both in control conditions and in response to salt stress, and that overexpression of EXPA11 improves salinity tolerance by increasing shoot growth under saline conditions without causing a growth penalty under control conditions. Overall, this study presents evidence that NIT2 likely affects both ion homeostasis and plant growth through the auxin-induced activation of the PM H+-ATPase, and downstream growth modulating activity of EXPA11, during salt stress.
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    Open Access
    Nitrilase 2: insight into its regulation in Arabidopsis and its potential for improving maize salt tolerance
    (2023) Foreman, Nina-Courtney; Donaldson, Lara; Ingle Robert
    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.