OpenUCT :: Browsing by Author "Donaldson, Lara"

Browsing by Author "Donaldson, Lara"

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Open Access
The arabidopsis cyclic nucleotide interactome
(BioMed Central, 2016-05-11) Donaldson, Lara; Meier, Stuart; Gehring, Christoph
Background: Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotidedependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods: An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results: A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions: We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.
Open Access
Characterisation of auxin and auxin-related genes in the response of Arabidopsis thaliana to salt stress
(2019) Cackett, Lee; Donaldson, Lara; Ingle, Robert
Soil salinization is prominent in agricultural land and has detrimental effects on plant growth and yield. Salt imposes both an osmotic and ionic stress on plants, and the aim of this study was to investigate the molecular basis of the plant response to the ionic component of salinity stress. To do so, transcriptome profiling using microarrays was performed at two developmental stages (two and four weeks post-germination) exposing plants to either NaCl (which imposes an osmotic and ionic stress) or iso-osmolar sorbitol (which imposes osmotic stress only). Clear transcriptomic differences in the plant response to NaCl and sorbitol were observed, allowing the identification of genes that may be involved specifically in the response to the ionic component of salinity stress. Differences in salt-responsive gene expression were also observed between early and later development as well as root and shoot tissues, indicating that there may be both developmental and tissue specific responses to NaCl. ‘Response to auxin’ was a highly enriched gene ontology term associated with genes that are significantly induced specifically in response to ionic stress, suggesting a potential role for this plant growth hormone in ionic stress tolerance. This hypothesis was supported by mass spectrometry analysis which demonstrated that active IAA levels show a significantly greater increase in response to NaCl than to sorbitol, demonstrating an ionic specific auxin response. In planta quantification and spatial distribution of IAA was further analysed in salt stressed plants using two auxin reporter lines, DR5::GUS and DII::VENUS. These analyses showed that IAA levels increased in the shoot and root tip in response to NaCl. Also, local IAA maxima distributed along the primary root of salt stressed seedlings were decreased compared to the untreated control, which may explain the decrease in lateral root number and primary root bending observed in salt stressed plants. There are several different pathways in the tryptophandependent auxin biosynthesis process, each with unique biosynthetic intermediates and ratelimiting enzymes. For this reason, changes in the levels of the different IAA biosynthetic intermediates were analysed in NaCl and sorbitol stressed Arabidopsis to identify a predominant pathway responsible for increasing IAA during ionic stress. The changes in IAN (the IAA biosynthetic intermediate of the IAOx pathway) in response to NaCl and sorbitol mirrored those observed for IAA. Additionally, mRNA levels of Nitrilase 2 (which converts IAN to IAA) were significantly increased in response to NaCl but not sorbitol. Taken together, these results suggest that IAA biosynthesis is increased in response to NaCl via the hydrolysis of IAN to IAA by NIT2. In agreement with this, a Nit2 overexpressing line (35s::Nit2) displayed both improved survival and growth in the presence of NaCl. Furthermore, the Nit2 overexpressor had increased IAA but deceased IAN compared to wild type plants in response to salt stress. Together these results indicate that Nit2 is likely involved in the salt stress response through altering IAA levels in planta. GH3.12 and ILL6, two genes involved in IAA storage and/or degradation via hydrolysis and conjugation to amino acids, were also differentially expressed in response to NaCl at both developmental stages tested. However, mutants for ill6 and gh3.12 were not altered in their salt tolerance, suggesting that IAA storage and/or degradation may not play an important role in modulating active IAA levels in response to salt stress. Finally, preliminary evidence was obtained that the expression of sorghum Nit2 homologs is also increased in response to salt stress, implying that auxin might play a role in salt tolerance across a phylogenetically broad range of plants, making this a potential route to improve salt tolerance in crop plants. Overall, this study provides novel information indicating that IAA levels are altered specifically in response to the ionic component of salinity stress which may contribute to the altered plant growth observed under these conditions, as well as the identification of an auxin-related candidate gene which can improve salt tolerance in plants when overexpressed.
Open Access
Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides
(BioMed Central Ltd, 2008) Meier, Stuart; Bastian, Rene; Donaldson, Lara; Murray, Shane; Bajic, Vladimir; Gehring, Chris
BACKGROUND: Plant natriuretic peptides (PNPs) are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A. RESULTS: A gene ontology analysis of AtPNP-A and the 25 most expression correlated genes revealed a significant over representation of genes annotated as part of the systemic acquired resistance (SAR) pathway. Transcription of these genes is strongly induced in response to salicylic acid (SA) and its functional synthetic analogue benzothiadiazole S-methylester (BTH), a number of biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore, the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70 lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and PR-5, were determined to be strongly induced. CONCLUSION: The co-expression analyses, both in wild type and mutants, provides compelling evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with PR proteins in that its transcription is strongly induced in response to pathogen challenges, it contains an N-terminal signalling peptide and is secreted into the extracellular space and along with PR-1, PR-2 and PR-5 proteins it has been isolated from the Arabidopsis apoplast. Based on these findings we suggest that AtPNP-A could be classified as a newly identified PR protein.
Open Access
Comparative analysis of Panicum streak virus and Maize streak virus diversity, recombination patterns and phylogeography
(BioMed Central Ltd, 2009) Varsani, Arvind; Monjane, Aderito; Donaldson, Lara; Oluwafemi, Sunday; Zinga, Innocent; Komba, Ephrem; Plakoutene, Didier; Mandakombo, Noella; Mboukoulida, Joseph; Semballa, Silla; Briddon, Rob; Markham, Peter; Lett, Jean-Michel; Lefeuvre, Pierre; Ry
BACKGROUND: Panicum streak virus (PanSV; Family Geminiviridae; Genus Mastrevirus) is a close relative of Maize streak virus (MSV), the most serious viral threat to maize production in Africa. PanSV and MSV have the same leafhopper vector species, largely overlapping natural host ranges and similar geographical distributions across Africa and its associated Indian Ocean Islands. Unlike MSV, however, PanSV has no known economic relevance. RESULTS: Here we report on 16 new PanSV full genome sequences sampled throughout Africa and use these together with others in public databases to reveal that PanSV and MSV populations in general share very similar patterns of genetic exchange and geographically structured diversity. A potentially important difference between the species, however, is that the movement of MSV strains throughout Africa is apparently less constrained than that of PanSV strains. Interestingly the MSV-A strain which causes maize streak disease is apparently the most mobile of all the PanSV and MSV strains investigated. CONCLUSION: We therefore hypothesize that the generally increased mobility of MSV relative to other closely related species such as PanSV, may have been an important evolutionary step in the eventual emergence of MSV-A as a serious agricultural pathogen.The GenBank accession numbers for the sequences reported in this paper are GQ415386-GQ415401
Open Access
Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts
(BioMed Central Ltd, 2009) Harkins, Gordon; Delport, Wayne; Duffy, Siobain; Wood, Natasha; Monjane, Aderito; Owor, Betty; Donaldson, Lara; Saumtally, Salem; Triton, Guy; Briddon, Rob; Shepherd, Dionne; Rybicki, Edward; Martin, Darren; Varsani, Arvind
BACKGROUND:Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. RESULTS: We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 x 10-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Reunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. CONCLUSION: The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.
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.
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.
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A novel species of mastrevirus (family Geminiviridae) isolated from Digitaria didactyla grass from Australia
(Springer Verlag, 2010) Briddon, Rob W; Martin, Darren P; Owor, Betty E; Donaldson, Lara; Markham, Peter G; Varsani, Arvind; Greber, Ray S
Mastreviruses (family Geminiviridae) that infect monocotyledonous plants occur throughout the temperate and tropical regions of Asia, Africa, Europe and Australia. Despite the identification of a very diverse array of mastrevirus species whose members infect African monocots, few such species have been discovered in other parts of the world. For example, the sequence of only a single monocot-infecting mastrevirus, Chloris striate mosaic virus (CSMV), has been reported so far from Australia, even though earlier biological and serological studies suggested that other distinct mastreviruses were present. Here, we have obtained the complete nucleotide sequence of a virus from the grass Digitaria didactyla originating from Australia. Analysis of the sequence shows the virus to be a typical mastrevirus, with four open reading frames, two in each orientation, separated by two noncoding intergenic regions. Although it showed the highest levels of sequence identity to CSMV (68.7%), their sequences are sufficiently diverse for the virus to be considered a member of a new species in the genus Mastrevirus, based on the present species demarcation criteria. We propose that the name first used during the 1980s be used for this species, Digitaria didactyla striate mosaic virus (DDSMV).

Browsing by Author "Donaldson, Lara"

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