Pachycladon species evolved traits to adapt to New Zealand habitats : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand

Figures are re-used with permission.The supplementary files in Appendix 2 may be made available on request to the Library.P. cheesemanii is a close relative of A. thaliana and is an allotetraploid perennial herb that is widespread in the South Island of New Zealand. It grows at altitudes of up to 1,000 m where it is subject to relatively high levels of UV-B radiation. However, to date the origin of this species and the mechanisms underlying its tolerance to its harsh living environmental conditions such as moderate–high UV-B radiation, cold and drought is unclear. To gain the first insights into how Pachycladon copes with UV-B stress, I sequenced the P. cheesemanii genome and compared the UV-B tolerance of plants from Wye Creek (~300-m altitude) and Kingston (~500-m altitude) with that of A. thaliana from Col-0 (~100-m altitude) and Kondara (1,000–1,100-m altitude). A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologues. A synteny analysis involving genomes from other species of the Brassicaceae family suggested that the two subgenomes of P. cheesemanii may have the same origin as species from Brassicaceae Lineage I and EII. While UV-B radiation caused greater growth reduction in A. thaliana Col-0 and Kondara than in P. cheesemanii Wye Creek, growth was not reduced in P. cheesemanii Kingston. Homologues of the A. thaliana UV-B radiation response genes have multiple copies in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii Wye Creek and Kingston may differ from that in A. thaliana. Although the P. cheesemanii genome shows close similarity with that of A. thaliana, the uniqueness of the strongly UV-B-induced UVR8-independent pathway in P. cheesemanii may help this species to tolerate relatively high UV-B radiation. Next, to understand the different stress responses of A. thaliana and P. cheesemanii, I designed a project to build multiple-stress transcriptomes for A. thaliana and P. cheesemanii. Since plant responses to salt and drought are related and have overlapping mechanisms, and salt stress can easily be applied in the laboratory, high salinity rather than drought stress was used to stress A. thaliana and P. cheesemanii plants in this study. Transcriptomes of A. thaliana and P. cheesemanii plants in response to cold, salt and UV-B radiation stresses were created. A high-quality de novo transcriptome assembly of allopolyploid P. cheesemanii was obtained by using multiple assemblers with further downstream processing. Differential expression analysis revealed a strong bias, in terms of the number of DEGs, towards upregulation in both A. thaliana and P. cheesemanii in responding to salt stress, as well as in P. cheesemanii’s cold and UV-B treatment responses. Meanwhile, in each species, a number of DEGs was shared between stresses, although the majority were unique in responding to each stress in upregulation and downregulation, respectively. Further, GO enrichment analysis revealed that these responsive genes were involved in some biological processes shared by A. thaliana and P. cheesemanii. Immune system processes, response to stimuli, signalling, developmental processes, growth, negative regulation of biological processes, multi-organism processes, biological regulation, secondary metabolic processes, cell communication, and cellular aromatic compound metabolic processes were common in the responses of both A. thaliana and P. cheesemanii to all three stresses. In both A. thaliana and P. cheesemanii, a number of these biological processes were also stress specific. First of all, in A. thaliana, cold stress may easily affect photomorphogenesis in cold responses, while the majority of the P. cheesemanii unique cold responses occurred in root differentiation, floral whorl development and regulation of programmed cell death. Second, A. thaliana responses to salt stress affected starch metabolism and lipid modification, whereas disaccharide and polysaccharide metabolism, as well as microtubule structure, were affected by salt stress uniquely in P. cheesemanii. Finally, A. thaliana responses to UV-B radiation involved a combination of physical and biological defences, including cell wall modification defence, stomatal movement, vitamin B6 metabolic processes and oxygen metabolic processes. In contrast, seed germination biological regulation was affected in P. cheesemanii under UV-B radiation stress. Further, P. cheesemanii had a larger number of unique GO enrichments in cold responses than did A. thaliana. There was a wide range of crosstalk among the biological processes in responding to the three stresses in A. thaliana, while only one main cluster was identified in crosstalk for the three stress responses in P. cheesemanii. In this main cluster, the biosynthetic process for anthocyanins was in the centre position, and it was found that multiple stress-responsive biological processes probably involved anthocyanins in P. cheesemanii. Thus, although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies such as a highly UV-B-activated UVR8-independent pathway, allowing the plant to tolerate relatively high UV-B radiation. The stress process is highly conserved in plant species under various stresses, but species also develop a few unique characteristics that may help them adapt to their own ecological niche and survive particular environmental stresses

Regulation of desiccation tolerance in Xerophyta seedlings and leaves

A small, diverse group of angiosperms known as resurrection plants display vegetative desiccation tolerance and can survive loss of up to 95% of cellular water, a feat only seen in the seeds and pollen of other angiosperms. Xerophyta humilis is a resurrection plant native to Southern Africa that has been the target of previous transcriptomic and proteomic studies into the mechanisms of plant desiccation tolerance. The aim of this study was to investigate the hypothesis that vegetative desiccation tolerance is derived from the networks that control desiccation tolerance in seeds and germinating seedlings in angiosperms, particularly the epigenetically silenced seed maturation genes. Germinating seedlings of X. humilis and the related resurrection plant X. viscosa were found to be VDT from the earliest stages of germination, and exhibited the characteristic vegetative trait of poikilochlorophylly as seen in mature leaves. The X. humilis desiccation transcriptome comprising 76,768 distinct gene clusters was successfully assembled from sequencing samples at five relative water contents (100%, 80%, 60%, 40% and 5%) to identify the networks activated in response to water loss. Desiccation was associated with successive waves of transcription factor induction, as well as widespread down-regulation of histone modification enzymes. Many seed-specific genes, such as late embryogenesis abundant (LEA) proteins, seed storage proteins and oleosins, were induced in vegetative tissue. LEA transcripts in particular were highly up-regulated during desiccation, and the large number of distinct LEA transcripts (over 150) suggests possible LEA gene expansion in Xerophyta compared to desiccation-sensitive plants. Components of the PYL/SnRK2/ABF ABA-signalling pathway were also induced, although the ABF transcription factors activated in response to desiccation were most similar to those induced by drought in A. thaliana rather than seed maturation. Of the canonical seed master regulators (such as the LEC1/ABI3/FUS3/LEC2 network and ABI5) only three ABI3 transcripts were expressed, all of which encoded proteins lacking the seed motif-binding B3-domain. The results of this study suggest that vegetative desiccation tolerance in X. humilis is not associated with re-activation of seed master regulators in vegetative tissue, but may instead involve activation of seed genes by vegetative drought response regulators

Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles

The papers included in this Special Issue address a variety of important aspects of Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles, including new advances in the sequencing of both mitochondria and chloroplasts’ genomes using Next-Generation Sequencing technology in plant species and algae including important crop and tree species, in vitro culture protocol, and identification of a core module of genes involved in plastid development. In particular, the published studies focus on the description of adaptive evolution, elucidate mitochondrial mRNA processing, highlight the effect of domestication process on plastome variability and report the development of molecular markers. A meta-analysis of recently published genome-wide expression studies allowed the identification of novel nuclear genes, involved in the complex and still unrevealed mechanisms at the basis of communication between chloroplast and nucleus (retrograde signalling) during plastid development (biogenic control). Finally, an optimized regeneration protocol useful in plastid transformation of recalcitrant species, such as sugarcane, has been reported

Regulation of chromatin positioning in Arabidopsis thaliana

The biochemical environment within the 3D nuclear space is not homogeneous. It has been demonstrated in many studies that the transcriptional activity of a gene is linked to its positioning inside the nuclear space. The NE not only serves as a physical barrier separating the nuclear content from the cytoplasm but also plays crucial roles in mediating the 3D organization of genomic DNA. Following the discovery of LADs, which are transcriptionally repressed chromatin regions, the non-random chromatin positioning at the NP and its biological relevance have been studied intensively in animals. However, it still remains unknown in plants that whether comparable chromatin organizations exist or not. In this study, RE-ChIP was used to reveal the genome-wide identification of non-random organization of chromatin domains positioned at the peripheral zone of Arabidopsis thaliana nuclei. The patterns of chromatin regions positioned at NP were similar across different tissues. These chromatin domains are enriched with silenced protein-coding genes, TE genes and heterochromatic marks, which collectively define a repressed environment at the NP. Furthermore, our results suggest a spatial compartment of different DNA methylation pathways that regulate TE silencing, where the CHH DNA methylation of TEs localized at the NP and in the nuclear interior is preferentially mediated by CMT2 and DRM methyltransferases, respectively. To elucidate how such chromatin positioning patterns at the NP was achieved in plants, dual-color FISH experiments were conducted to compare the difference of chromatin-NP interactions among various mutants. Our results show that in Arabidopsis thaliana, specific chromatin positioning at the NP requires plant lamin proteins CRWN1, CRWN4 and non-CG DNA methylation, which are all plant-specific. The result of chromosome painting indicates global attenuation of chromatin positioning patterns at the NP in both the crwn1 and crwn4 mutants. Moreover, ChIP-seq shows that CRWN1 directly interacts with chromatin regions localized at the NP. In summary, the NP is a functional sub-compartment enriched with heterochromatic domains. In addition, CRWN1 is a key component of lamin-chromatin network in plants. It is functionally equivalent to animals lamins, which play crucial roles in regulating chromatin positioning at the NP

Long non-coding RNAs in siliques of Arabidopsis thaliana ecotypes and PRC2 mutants

Transcriptomic studies from many eukaryotic species have shown that in addition to protein coding mRNA, there exists RNA that appears to have no protein coding potential. One class of such RNAs are long non-coding RNAs (lncRNAs) whose length is greater than 200 nucleotides. In this research project, the extent and diversity of lncRNAs expressed during developing siliques was investigated. To achieve this, next-generation RNA-sequencing (Illumina®) was performed on Arabidopsis thaliana silique RNA for ecotypes Col-0 and C24. Reciprocal crosses of these two ecotypes were also sequenced, to investigate potential parent of origin expression in developing seeds of these siliques. After assembling transcripts, known gene models and transcripts containing peptide potential were removed, revealing 2,807 potential lncRNAs. The lncRNAs identified had diverse genomic locations; antisense to protein coding mRNAs, sense and antisense within both intergenic regions and intronic regions. LncRNAs had a median length of approximately 500 nt, contained one to two exons but minorities were alternatively spliced. Candidate lncRNAs were investigated, some being potentially imprinted, others being in proximity to genes expressed exclusively in the endosperm and a minority of lncRNAs were methylated. In animals and plants, it is known that lncRNAs bind to Polycomb Repressive Complex 2 (PRC2) and are located at loci targeted by PRC2. To further investigate this in silique and seed development, lncRNAs were identified in reproductive-specific PRC2 mutants. A total 2,362 lncRNAs were identified; 55% (1,296) were exclusively identified in PRC2 mutants and are potentially targeted by PRC2. PRC2 mutants induced transcriptome wide differential expression of 8,212 genes, in particular transcriptional regulators, transcription factors and DNA binding proteins. Furthermore, 520 lncRNAs were differentially expressed in PRC2 mutants. Novel lncRNA candidates were explored, many being exclusively expressed in the absence of PRC2 and were in proximity to key genes involved in transcription regulation, such as transcription factors. As PRC2 regulates endosperm development and governs post-zygotic hybridisation barriers, inter-genus crosses between Arabidopsis thaliana and Boechera pinetorum created with mutations in PRC2 were investigated as part of a long term aim. Using PCR and genotyping sequencing, it was confirmed that all hybrids contained the genomes of both distant parents. It was confirmed that PRC2 mutations facilitated the generation of A. thaliana x Boechera pinetorum hybrids, although it is not known how. With the hybrids confirmed, this provided a platform for research into roles of lncRNAs in alleviating post-zygotic hybridisation barriers. Overall, this research project identified a total 4,147 lncRNAs in A. thaliana siliques from various ecotypes, crosses and mutants. 64% (2,652) were novel, not being reported by any other study. However, further experiments are required to validate lncRNAs and elucidate their functions. The 4,147 lncRNAs identified are an important contribution to plant lncRNA research, providing a novel resource to understand the role lncRNAs play in plant biology

Jasmonic Acid Pathway in Plants

The plant hormone jasmonic acid (JA) and its derivative, an amino acid conjugate of JA (jasmonoyl isoleucine, JA-Ile), are signaling compounds involved in the regulation of defense and development in plants. The number of articles studying on JA has dramatically increased since the 1990s. JA is recognized as a stress hormone that regulates the plant response to biotic stresses such as herbivore and pathogen attacks, as well as abiotic stresses such as wounding and ultraviolet radiation. Recent studies have remarkably progressed the understanding of the importance of JA in the life cycle of plants. JA is directly involved in many physiological processes, including stamen growth, senescence, and root growth. JA regulates production of various metabolites such as phytoalexins and terpenoids. Many regulatory proteins involved in JA signaling have been identified by screening for Arabidopsis mutants. However, much more remains to be learned about JA signaling in other plant species. This Special Issue, “Jasmonic Acid Pathway in Plants”, contains 5 review and 15 research articles published by field experts. These articles will help with understanding the crucial roles of JA in its response to the several environmental stresses and development in plants

Genetic analysis of developmental traits associated with enhanced winter survival in autumn-seeded rye (Secale cereale L.).

The abstract of this item is unavailable due to an embargo

Unravelling the Complexity of the Molecular and Physiological Response to Environmental Change in Seagrasses

This thesis explores the complexity of seagrass stress response in the face of current environmental changes. This is a timely and relevant issue due to the role supplied by these foundation species in coastal ecosystems, and the dramatic consequences their loss would cause on marine biodiversity and human well-being. Using as target species the iconic Mediterranean seagrass Posidonia oceanica, here I show: i) how molecular reprogramming, acting primarily at gene-expression level, coordinates physiological and morphological responses to different stressors, and ultimately determines species’ acclimation strategies and tolerance capacity; ii) the differential stress response existing within and among different organs, and between different shoot types; iii) how the response to a single stressor can be modified depending on its temporal variability, and due to the interaction with another stressor. In this study, new transcriptome data have been generated, from leaves and shoot-apical meristems, increasing considerably molecular resources available for future studies on seagrass evolutionary ecology and functional genomics. Moreover, this research sheds first light on the stress response of organs other than leaf, in seagrasses, and recognises the shoot meristem as a key determinant of whole plant survival. Common and stress-specific molecular biomarkers have been identified through different approaches, and their potential applicability as sub-lethal stress indicators can be verified in the future with ad hoc experiments. Another important aspect of this study is the recognition of the importance of epigenetic variations, specifically DNA methylation changes, as key mechanisms for phenotypic accommodation and adaptive responses to environmental changes in seagrasses. Tolerance capacity of the species to main current threats of coastal areas, namely the reduction of available light, heat stress, eutrophication and herbivory, is discussed in light of the results obtained from the different experiments