M.Sc. / Lipopolysaccharides (LPS) are ubiquitous, indispensable components of the cell surface of Gram-negative bacteria that have diverse roles in bacterial pathogenesis of plants. LPS as pathogen-associated molecular pattern (PAMP) molecules can be recognized by plants to directly trigger some defense—related responses. LPS can also alter the response of plants to subsequent bacterial inoculation; these delayed effects include alterations in the expression patterns of genes coding for some pathogenesis related (PR) proteins, promotion of the synthesis of the antimicrobial conjugates, and prevention of the hypersensitive reaction caused by avirulent bacteria. Prevention of the response may allow expression of resistance in the absence of catastrophic tissue damage. LPS from Burkholderia cepacia (LPSB. cep.) have been found to trigger a strong response in plants resulting in the activation of genes coding for some pathogenesis related proteins, receptor-like kinases and resistance (R) proteins. LPS are tripartite amphipathic molecules, consisting of a Lipid A moiety that is embedded in the outer leaflet of the phospholipids/protein bilayer, a core oligosaccharide, and a polysaccharide consisting of repeating units (0-Antigen/O-side chain). Typically the Lipid A consists of a bisphosphorylated glucosamine disaccharide which is substituted by amide- and ester-bound fatty acids and / or acyloxyacyl groups. The core region, a non-repetitive oligosaccharide, is usually connected to the Lipid A part via one 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residue. The core is attached in turn to the 0- Antigen that consists in most cases of a repetitive polysaccharide and that represents the major part of LPS. The bond between the Lipid A section and the Kdo residue of the core is labile under mild acid hydroysis conditions; and this allows for the fractionation of the LPS molecule into a Lipid A part and an 0-Antigen part, attached to the core. Thus far the eliciting (active) parts of LPSB. cep. have not yet been identified. In general, it is known that the Lipid A is more conserved from one organism to another as compared to the 0-Antigen. In animals, Lipid A is believed to be the active part as it was found to elicit some defense-related responses. In plants, Lipid A was also found to trigger defense responses. Several structures of the 0-Antigens from different bacteria have been characterised, but their biological activities have not yet been investigated in detail.

Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering iii of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.

Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing up to 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expression during sugarcane growth and maturation is limited. The aim of this study was to assess whether an Expressed Sequence Tag (EST)-based approach towards analysis of sugarcane would reveal new information about gene expression and metabolic processes associated with sugarcane growth and development. The specific objectives were two-fold: firstly, to develop an EST database for sugarcane and secondly, to identify and analyse genes that are expressed in different sugarcane tissue types and developmental stages, with a specific focus on leaf and culm. An EST database for sugarcane was initiated to obtain information on sugarcane gene sequences. A total cDNA library was constructed from sugarcane immature leaf (leaf roll: meristematic region) tissue and 250 clones randomly selected and subjected to single-pass DNA sequence analysis. Sugarcane ESTs were identified by sequence similarity searches against gene sequences in international databases. Of the 250 leaf roll clones, 26% exhibited similarity to known plant genes, 50% to non-plant genes while 24% represented new gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes. A significant proportion of genes identified in the leaf roll were involved in processes related to protein synthesis and protein modification, as would be expected in meristematic tissues. Submission of 495 sugarcane gene sequences to the dbEST database represented the first sugarcane ESTs released into the public domain. Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature and maturing internodal tissue. To explore gene expression during sugarcane culm maturation, partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries was performed. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases while 111 matched uncharacterised plant ESTs only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. The expression patterns of sugarcane genes were examined in different tissue sources and developmental stages to identify differentially expressed genes. cDNA arrays containing 1000 random clones from immature leaf and maturing culm cDNA libraries were hybridised with poly (At RNA from immature leaf, mature leaf, immature culm and maturing culm. All cDNAs examined hybridised to all four probes, but differences in signal intensity were observed for individual cDNAs between hybridisation events. No cDNAs displaying tissue- or developmental-stage specific expression were detected. Comparisons between hybridisation patterns identified 61 cDNAs that were more abundantly expressed in immature and mature leaf than the culm. Likewise, 25 cDNAs preferentially expressed in immature and maturing culm were detected. ESTs established for the differentially expressed cDNAs revealed sequence homology to a diverse collection of genes in both the leaf and the culm. These included genes associated with general cellular metabolism, transport, regulation and a variety of stress responses. None of the differentially expressed genes identified in the culm were homologous to genes known to be associated with sucrose accumulation. To examme differences at the level of gene transcription between low sucroseaccumulating and high sucrose-accumulating tissues, subtracted cDNA libraries were utilised. To isolate cDNAs differentially expressed during culm maturation, cDNA arrays containing 400 random clones (200 from each library) were screened with total cDNA probes prepared from immature and maturing culm poly (At RNA. Results indicated that 36% and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-preferential expression for most of the clones tested. ESTs generated for the 132 differentially expressed clones isolated exhibited homology to genes associated with cell wall metabolism, carbohydrate metabolism, stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. These results suggest that growth and maturation of the sugarcane culm is associated with the expression of genes for a wide variety of metabolic processes. In addition, genes encoding enzymes directly involved with sucrose accumulation do not appear to be abundantly expressed in the culm. / AFRIKAANSE OPSOMMING: Kommersiële suikerriet variëteite (Saccharum spp. hibriede) is in staat om tot 20% sukrose op 'n vars massa basis in die stingel op te berg. Kennis oor geenuitdrukking tydens groei en rypwording is beperk. Die doel van die huidige studie was om vas te stelof 'n grootskaalse karatersisering van die geenvolgordes wat uitgedruk word "Expressed Sequence Tag (EST)-based approach" tot nuwe inligting aangaande die aard en omvang van metabolisme tydens groei en ontwikkeling van suikerriet sal lei. 'n Tweeledige benadering is in hierdie studie gevolg. Eerstens is 'n data basis oor die gene wat uitgedruk word "EST" databasis opgestel. Tweedens is gene geïdentifiseer en gekarakteriseer wat spesifiek op verskillende stadiums van ontwikkeling en in spesifiek weefsel uitgedruk word. Vir die opstel van die EST-databasis is 250 klone uit 'n totale cDNA biblioteek vanaf RNA uit suikerrietblaarweefsel (blaarrol:meristematiese streek) op 'n lukraak basis gekies en aan 'n enkel eenrigting DNA volgorde analise onderwerp. Suikerrriet EST's is geïdentifiseer deur middel van homologie soektogte teen geenvolgordes in internasionale databasisse. Uit die 250 blaarrol klone het 26% ooreenkomste met bekende plant gene en, 50% met nie-plant gene getoon. Ongeveer 24% het nuwe geenvolgordes verteenwoordig. Analise van die geïdentifeseerde klone het ooreenkomste met 'n breë diversiteit van gene getoon. 'n Betekenisvolle gedeelte van gene wat in die blaarrol geïdentifiseer is, is by proteïensintese en proteïenmodifikasies betrokke. Dit is in ooreenstemming met wat van meristematiese weefsel verwag kan word. Die 495 suikerriet geenvolgordes wat in die internasionale dbEST databasis gestort is, is die eerste sodanige inligting in die publieke domein. Twee spesifieke cDNA biblioteke (subtraction libraries) wat volgordes spesifiek aan onvolwasse suikerriet en rypwordende internodale weefsel bevat is voorberei. Geenuitdrukking gedurende die rypwordingsproses van die suikerrietstingel is bestudeer deur geenvolgorde analises van onwillekeurige geselekteerde klone van die twee eDNA biblioteke te doen. Van die 337 geenvolgordes wat geanaliseer is het 167 homologie met bekende gene en net 111ooreenkomste met ongekarakteriseerde plant gene getoon. Die oorblywende geenvolgordes het geen ooreenkomste met bekende gene getoon nie en daar kan dus aanvaar word dat hulle nuwe gene verteenwoordig. Die meerderheid ESTs het ooreenkomste met verskeie gene wat met sellulêre metabolisme geassosieer word getoon. ESTs wat homoloog was aan verskeie spannings geassosieerde gene was ook goed verteenwoordig. Die analise het gene wat by voorkeur tydens stringelrypwording uitgedruk word geidentifiseer. Die geenuitdrukkingspatrone van suikerriet in weefsels van verskillende oorsprong en ontwikkelingstadia is ondersoek om differensieel uitgedrukte gene te identifiseer. Reekse wat 1000 lukrake eDNA klone van onvolwasse en rypwordende stingel eDNA biblioteke is met poli-(A)-RNA van onvolwasse blaar, volwasse blaar, onvolwasse stingel en volwasse stingel gehibridiseer. Al die eDNA klone wat ondersoek is het met al vier die peilers gehibridiseer. Die intensiteit van die seine het egter grootliks gevarieer. Die analise het gelei tot die identifisering van 61 eDNA klone wat teen hoër vlakke in onvolwasse en volwasse blaar as in die stingel uitgedruk word. Daar is ook 25 eDNA klone wat by voorkeur in onvolwasse en rypwordende stingel uitgedruk word gevind. Gene wat geassosieer word met gewone sel metabolisme, vervoer prosesse, regulering en verskeie spannings-geassosieerde reaksies, is in die twee groepe teenwoordig. Geeneen van die volgordes wat selektief uitgedruk word kan met gene wat direk met sukrose akkumulering verband hou geassosieer word nie. Ten einde eDNA klone wat differensieel tydens rypwording van die stingel uitgedruk word te isoleer, is 400 eDNA klone (200 van elke biblioteek) lukraak geselekteer en met totale eDNA peilers, wat uit onvolwasse en rypwordende stingel poli-(A)-RNA voorberei is, gesif. Resultate het aangetoon dat 36% en 30% van die totale getal eDNA klonewat geanaliseer is, by voorkeur in die onvolwasse en rypwordende stingel uitgedruk word. RNA kladanalises van geselekteerde klone het getoon dat die meeste ontwikkelingstadium spesifieke uirtdrukkingspatrone het. Daar is gevind dat 132 van die EST klone homologie met gene geassosieerd met selwand- en koolhidraatmetabolisme, spannings geassosieerde- en reguleringsreaksies, toon. Die spesifieke ESTs wat in die onvolwasse en rypwordende stingel geïdentifiseer is het van mekaar verskil. Nie een van die ESTs wat geïdentifiseer is kan direk met sukrose metabolisme geassosieer word nie. Hierdie werk toon baie duidelik aan dat groei en rypwording van die suikerrietstingel met die uitdrukking van gene geassosieerd is wat by 'n hele aantal metaboliese prosesse betrokke is. Die resultate toon ook dat die gene wat vir ensieme kodeer wat direk by sukrose akkumulering betrokke is, nie teen hoë vlakke in die stingel uitgedruk word nie.

<p><i>Agrobacterium tumefaciens </i>transfers virulence effector proteins to infected host plants to facilitate the transfer and trafficking of a piece of its tumor inducing (Ti) plasmid, (T-[transfer] DNA), into and through plant cells.T-DNA integrates into the host genome where it uses the host’s gene expression machinery to express transgenes. Scientists have used this process to insert beneficial genes into plants by replacing native T-DNA in the bacteria with engineered T-DNA, making <i>Agrobacterium</i>-mediated transformation the preferred method for crop genetic engineering. In spite of its wide-spread use in research and agriculture, we still do not have a complete understanding of the transformation process. Consequently, many important crop species remain highly resistant to transformation. One of my lab’s major goals is to define the molecular interactions between <i>Agrobacterium</i> and its host plants which mediate transformation. I study the role of the <i>Agrobacterium</i> effector protein, VirE2, which is important for plant transformation. VirE2 likely coats the transferred DNA (T-DNA) after it enters the plant cell and protects it from degradation. VIP1 is a host transcription factor that interacts with VirE2 and is involved in activating plant defense responses. VIP1 localizes to both the cytoplasm and the nucleus.Under stress, VIP1 localizes to the nucleus where it activates expression of defense response genes.This observation led to the model that T-DNA-bound VirE2 binds VIP1 and uses VIP1 nuclear localization to deliver T-DNA into the nucleus (the “Trojan Horse” model). In contrast to this model, our lab has obtained data showing that VirE2 holds at least a portion of the VIP1 pool outside the nucleus. We also showed that VIP1 and its homologs are not necessary for transformation. VirE2 interacts with several host proteins in addition to VIP1, and these interactions could lead to changes in host gene expression and protein levels, possibly facilitating transformation. We investigated this model by placing VirE2 under the control of an inducible promoter in <i>Arabidopsis</i> and performing RNA-seq and proteomics under non-induced and induced conditions, and in the presence of <i>Agrobacterium</i> to determine its individual effect on plant RNA and protein levels during infection. Some genes differentially expressed after VirE2 induction are known to be important for transformation. Knockout mutant lines of some VirE2 differentially expressed genes showed altered transformation phenotypes. Protein levels of genes known to be important for transformation were also increased in response to VirE2 induction, and overexpression of some of these genes resulted in increased transformation susceptibility. We therefore conclude that VirE2 modulates both plant RNA and protein levels to facilitate transformation.</p>

Water scarcity induced by drought, temperature, and salinity has plagued agricultural sustainability in recent years with unprecedented revenue losses, raising concerns for worldwide food security. Recent studies have revealed unique botanical response mechanisms to combat water related stress, namely the expression of proteins known as the dehydrins. Dehydrin proteins have been shown to serve various intracellular protective functions. The gene for a SK5 type dehydrin from the arctic plant Cerastium arcticum (CaDHN) was introduced into tobacco plants and water deficit tolerance was evaluated. Plants overexpressing CaDHN displayed improved tolerance to salt stress, but no improvement was observed under drought stress. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Chan Yee-kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-131). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / Acknowledgments --- p.v / General Abbreviations --- p.vii / Abbreviation for Chemicals --- p.x / Table of Contents --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Salinity and agriculture --- p.2 / Chapter 1.3 --- Plant adaptation to salinity --- p.4 / Chapter 1.3.1 --- Salt secretion --- p.6 / Chapter 1.3.2 --- Ion transport --- p.8 / Chapter 1.3.2.1 --- Role of H+-ATPase in salt tolerance --- p.8 / Chapter 1.3.2.2 --- Potassium and sodium uptake --- p.13 / Chapter 1.3.2.3 --- Sodium efflux --- p.15 / Chapter 1.3.3 --- Osmotic adjustment --- p.20 / Chapter 1.3.3.1 --- Accumulation of mannitol --- p.21 / Chapter 1.3.3.2 --- Accumulation of proline --- p.23 / Chapter 1.3.3.3 --- Accumulation of glycinebetaine --- p.23 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Plant materials and growth conditions --- p.26 / Chapter 2.1.1 --- Surface sterilization of Arabidopsis seeds --- p.26 / Chapter 2.1.2 --- Determination of sub-lethal inhibitory doses of sodium --- p.27 / Chapter 2.1.3 --- Growth conditions of Arabidopsis seeds for total RNA extraction --- p.27 / Chapter 2.1.4 --- NaCl dosage tests --- p.28 / Chapter 2.1.5 --- Expression kinetic tests --- p.28 / Chapter 2.2 --- Isolation of total RNAs --- p.28 / Chapter 2.3 --- Isolation of genes differentially expressed in NaCl concentration by RAP-PCR --- p.30 / Chapter 2.3.1 --- RNA fingerprinting by RAP-PCR --- p.30 / Chapter 2.3.2 --- PCR reamplificatin of RAP products --- p.31 / Chapter 2.3.3 --- Cloning of differentially expressed genes --- p.33 / Chapter 2.3.3.1 --- Ligation of inserts into pCR-Script vector and transformation --- p.33 / Chapter 2.3.3.2 --- Ligation of inserts into pBluescript II KS (+) T-vector and transformation --- p.36 / Chapter 2.3.3.3 --- Screening of recombinant plasmids --- p.37 / Chapter 2.4 --- Sequencing of differentially expressed genes --- p.39 / Chapter 2.4.1 --- DNA cycle sequencing --- p.39 / Chapter 2.5 --- Northern blot hybridization of NaCl inducible genes --- p.40 / Chapter 2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.40 / Chapter 2.5.2 --- Northern blotting --- p.41 / Chapter 2.5.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.41 / Chapter 2.5.3.1 --- Isolation of Total RNA --- p.41 / Chapter 2.5.3.2 --- Primer design --- p.42 / Chapter 2.5.3.3 --- PCR amplification of single-stranded DIG PCR probes --- p.43 / Chapter 2.5.4 --- Hybridization --- p.45 / Chapter 2.5.5 --- Stringency washes --- p.46 / Chapter 2.5.6 --- Chemiluminescent detection --- p.46 / Chapter 3. --- Results / Chapter 3.1 --- Determination of sub-lethal inhibitory doses of sodium --- p.48 / Chapter 3.2 --- Isolation of total RNA from A. thaliana treated with sodium chloride --- p.48 / Chapter 3.3 --- Isolation of genes differentially expressed in sodium concentration by RNA arbitrarily primed polymerase chain reaction RAP-PCR --- p.52 / Chapter 3.3.1 --- Differential cDNA fragments identified by RAP-PCR --- p.52 / Chapter 3.3.2 --- PCR reamplification of RAP products --- p.52 / Chapter 3.3.3 --- Cloning of selected RAP-fragments --- p.62 / Chapter 3.4 --- Nucleotide sequence analysis of selected RAP PCR clones --- p.65 / Chapter 3.5 --- Expression pattern analysis of salt inducible genes by northern blot hybridization --- p.75 / Chapter 3.5.1 --- Preparation of single-stranded digoxigenin (DIG)-labeled probes --- p.75 / Chapter 3.5.2 --- Dosage response of NaCl inducible genes --- p.79 / Chapter 3.5.3 --- Expression kinetics of NaCl inducible genes --- p.80 / Chapter 4. --- Discussion / Chapter 4.1 --- Isolation of RAP-PCR targets --- p.93 / Chapter 4.2 --- Expression of NaCl inducible P450 genes --- p.94 / Chapter 4.2.1 --- Cytochrome P450 CYP73A5 --- p.97 / Chapter 4.2.2 --- Cytochrome P450 CYP83A1 --- p.98 / Chapter 4.3 --- NaCl induction gene related to post-transcriptional activities --- p.99 / Chapter 4.3.1 --- Glycine-rich RNA binding protein (BAC F3F19) --- p.100 / Chapter 4.3.2 --- Chloroplast signal recognition particle (54CP) --- p.103 / Chapter 4.4 --- Conclusion --- p.106 / References --- p.108

No description available.

Previous studies have shown that the mitochondrial orf224/ atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus, and that the effects of nuclear fertility restoration on orf224/ atp6 transcripts co-segregate with the pol restorer gene Rfp in genetic crosses. Results presented in this thesis indicate that the pol CMS restorer gene Rfp acts in an organ-specific manner to promote the processing of primary, dicistronic orf224/atp6 transcripts into new restorer-specific, monocistronic transcripts. The single 1.1 kb atp6 transcript of nap cytoplasm and some orfB transcripts of nap and pol cytoplasm B. napus mitochondria are shown to arise by removal of sequences from the 5' end of a longer precursor. Specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may thus represent a common mechanism for tailoring transcripts in plant mitochondria. Northern analysis of a segregating F2 population indicates that the recessive rfp allele at the restorer gene locus, or a very tightly linked gene, acts as a dominant gene designated Mmt (modifier of mitochondrial transcripts). Mmt controls the presence of additional, smaller transcripts of the nad4 and ccl1-like (ccl1-l) genes. These results suggest that Rfp/Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes. One of these genes, ccl1-l, is split in the B. napus mitochondrial genome into two widely separated and independently transcribed parts, contrasting with the situation in other plants where this gene is present as an uninterrupted ORF. Although transcripts of both parts are edited, no "trans-spliced" transcripts spanning both parts of the split ORF, that could be translated into a full sized protein product, were detected. Moreover, an antiserum directed against the product of t

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008