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Item Comparative Transcriptomics of Multi-Stress Responses in Pachycladon cheesemanii and Arabidopsis thaliana.(MDPI (Basel, Switzerland), 2023-07-11) Dong Y; Gupta S; Wargent JJ; Putterill J; Macknight RC; Gechev TS; Mueller-Roeber B; Dijkwel PP; You FMThe environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii, grows under environmental conditions that are unsustainable for many plant species. Here, we compare the responses of both species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were determined and comparative transcriptome and network analyses discovered similar and unique responses within species, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a more important strategy to contribute to the UV-B radiation tolerance in P. cheesemanii. These results allowed us to define broad stress response pathways in A. thaliana and P. cheesemanii and suggested that regulation of glycosinolate, proline and anthocyanin metabolism are strategies that help mitigate environmental stress.Item The LONELY GUY gene family: from mosses to wheat, the key to the formation of active cytokinins in plants(John Wiley and Sons Ltd on behalf of the Society for Experimental Biology and The Association of Applied Biologists, 2022-04-07) Chen L; Jameson GB; Guo Y; Song J; Jameson PELONELY GUY (LOG) was first identified in a screen of rice mutants with defects in meristem maintenance. In plants, LOG codes for cytokinin riboside 5'-monophosphate phosphoribohydrolase, which converts inactive cytokinin nucleotides directly to the active free bases. Many enzymes with the PGGxGTxxE motif have been misannotated as lysine decarboxylases; conversely not all enzymes containing this motif are cytokinin-specific LOGs. As LOG mutants clearly impact yield in rice, we investigated the LOG gene family in bread wheat. By interrogating the wheat (Triticum aestivum) genome database, we show that wheat has multiple LOGs. The close alignment of TaLOG1, TaLOG2 and TaLOG6 with the X-ray structures of two functional Arabidopsis thaliana LOGs allows us to infer that the wheat LOGs 1-11 are functional LOGs. Using RNA-seq data sets, we assessed TaLOG expression across 70 tissue types, their responses to various stressors, the pattern of cis-regulatory elements (CREs) and intron/exon patterns. TaLOG gene family members are expressed variously across tissue types. When the TaLOG CREs are compared with those of the cytokinin dehydrogenases (CKX) and glucosyltransferases (CGT), there is close alignment of CREs between TaLOGs and TaCKXs reflecting the key role of CKX in maintaining cytokinin homeostasis. However, we suggest that the main homeostatic mechanism controlling cytokinin levels in response to biotic and abiotic challenge resides in the CGTs, rather than LOG or CKX. However, LOG transgenics and identified mutants in rice variously impact yield, providing interesting avenues for investigation in wheat.Item Developmentally controlled changes during Arabidopsis leaf development indicate causes for loss of stress tolerance with age(Oxford University Press on behalf of the Society for Experimental Biology, 2020-10-22) Kanojia A; Gupta S; Benina M; Fernie AR; Mueller-Roeber B; Gechev T; Dijkwel PP; Foyer CLeaf senescence is the final stage of leaf development and is induced by the gradual occurrence of age-related changes (ARCs). The process of leaf senescence has been well described, but the cellular events leading to this process are still poorly understood. By analysis of progressively ageing, but not yet senescing, Arabidopsis thaliana rosette leaves, we aimed to better understand processes occurring prior to the onset of senescence. Using gene expression analysis, we found that as leaves mature, genes responding to oxidative stress and genes involved in stress hormone biosynthesis and signalling were up-regulated. A decrease in primary metabolites that provide protection against oxidative stress was a possible explanation for the increased stress signature. The gene expression and metabolomics changes occurred concomitantly to a decrease in drought, salinity, and dark stress tolerance of individual leaves. Importantly, stress-related genes showed elevated expression in the early ageing mutant old5 and decreased expression in the delayed ageing mutant ore9. We propose that the decreased stress tolerance with age results from the occurrence of senescence-inducing ARCs that is integrated into the leaf developmental programme, and that this ensures a timely and certain death.Item Effector-triggered immunity against Pseudomonas syringae pv. actinidiae in nonhost plants : thesis submitted to the Massey University for the degree of Doctor of Philosophy(Massey University, 2017) Jayaraman, JayPseudomonas syringae pv. actinidiae (Psa) is a virulent and highly damaging pathogen causing bacterial canker in all currently commercially important cultivars of kiwifruit (Actinidia spp.). Arabidopsis and Nicotiana spp. plants, however, are nonhosts to Psa. In our course of investigating the various nonhost resistance mechanisms in play against Psa, we identified several sources of resistance against several Psa strains as well as a possible novel virulence mechanism used by Psa and Hyaloperonospora arabidopsidis (Hpa), a b iotrophic pathogen of Arabidopsis. Firstly, we discovered that the highly virulent strain, Psa V13, triggers hypersensitive response (HR) in Arabidopsis in an accession-‐specific manner and that HopZ5PsaV13, a member of the YopJ family of putative acetyltransferases, confers this bacterial avirulence. We also show that the immunity triggered by HopZ5 is independent from HR in the Arabidopsis accession Col-‐0. Through mutagenesis, we show that key amino acid residues predicted for acetyltransferase activity are vital to HopZ5-‐triggered immunity and HR, phenotypes reproduced in Nicotiana spp. Secondly, we identified multiple sources of avirulence for the kiwifruit low-‐ virulence strain, Psa LV5, in Arabidopsis and Nicotiana benthamiana, namely homologs of previously characterized effectors, HopAR1 and HopAB3, respectively. We additionally show that HopAB3 can trigger resistance in cultivated tomato putatively due to a novel recognition by a cultivated tomato homolog (SlPtoB) of the resistance gene Fen. Finally, we identified several nuclear-‐localized effectors from Psa and Hpa that interact with Arabidopsis WRKY transcription factors, different to WRKYs targeted by previously identified AvrRps4 and PopP2. We show that some WRKYs can trigger a cell death response in N. benthamiana when overexpressed and that coexpression of AvrRps4 or PopP2 is able to suppress this cell death response for the WRKYs they interact with. We show that this suppression is associated with suppression of transcriptional activation ability of the WRKY and 7 propose that this mechanism of transcription suppression may be utilized by other Psa and Hpa effectors identified in this study.Item Roles of a major O-acetylserine (thiol) lyase (OASTL) in cysteine biosynthesis, innate immunity and disease resistance in Arabidopsis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2012) Tahir, JibranO-Acetylserine (thiol) lyases (OASTLs) are evolutionary conserved proteins among many prokaryotes and eukaryotes that carry out sulphur acquisition and synthesis of cysteine. OASTL catalyses cysteine biosynthesis using O-acetylserine (OAS) and sulfide as substrates. OASTL also interact with another enzyme Serine acetyltransferase (SERAT) to facilitate the production of OAS. Cysteine-derived thiols and metabolites play an important function in regulating cellular redox conditions and modulate abiotic and biotic stress responses. The Arabidopsis thaliana genome encodes multiple OASTL isoforms that are targeted to different sub-cellular compartments. The cytosolic OASTL-A1 or known as ONSET OF LEAF DEATH3 (OLD3) is the major OASTL isoform due to its high OASTL activity. The old3-1 mutation causes a dysfunctional oastl-a1/old3-1 protein in vitro and was previously shown to cause autonecrosis in specific Arabidopsis accessions. To investigate why a mutation in a major OASTL isoform causes cell death and necrosis in some but not other accessions different mutations in OASTL-A1 were characterised in Arabidopsis accessions. Here it is shown that the old3-1 mutation causes an autoimmune syndrome and metabolic disorder, in the Ler-0 accession (parent) genetic background, but not in the reference accession Col-0. This is not the result of lack of functional OASTL-A1 or impaired cysteine biosynthesis. A Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene, from an evolutionary divergent R gene cluster in Ler-0, shows a negative epistatic interaction to old3-1 and activates autonecrosis. The severity of autonecrosis was found to be dependent upon variations in temperature and day length. Next, the role of OASTL-A1 was also identified in resistance against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. Since OASTL also interacts with SERAT, old3-1 was found to negatively affect the interaction with SERAT in vivo, highlighting that the release of R-mediated immunity is associated with the loss of key functions associated with OASTL. Finally various mutations were generated in OASTL-A1 isoforms to identify the relevance between the loss of OASTL functions and R-mediated immunity. These results indicated that motifs in close proximity of old3-1 mutation play an important role in cysteine biosynthesis and therefore likely an important interface to affect R-mediated immunity. The study indicates a novel cross-talk between cysteine procuring a major OASTL isoform and components of plant immunity and further support emerging evidence that cysteine-derived metabolites function in immune signalling across kingdoms.Item Within and between whorls: Comparative transcriptional profiling of Aquilegia and Arabidopsis(Public Library of Science, 2010) Voelckel C; Borevitz J; Kramer E; Hodges SBACKGROUND: The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels. METHODOLOGY/PRINCIPAL FINDINGS: We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource). CONCLUSIONS/SIGNIFICANCE: Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.