Auxinic herbicide-resistant (i.e., resistant to 2,4-D and MCPA) wild radish (Raphanus raphanistrum L.) was discovered in the Western Australian wheatbelt, providing an opportunity to integrate auxinic herbicide resistance into cultivated radish (R. sativus L.) using conventional breeding methods. It was hypothesized that the inheritance of auxinic herbicide resistance in wild radish is conferred by a single, dominant nuclear gene and, therefore, will be relatively easy to introgress from wild radish to cultivated radish; and the mechanism of auxinic herbicide resistance in wild radish is through an altered target-site. Visual injury data of the F2 progeny suggested that resistance was conferred by a quantitative trait with the susceptible allele(s) exhibiting dominance with minor cytoplasmically inherited genes masking the susceptible trait. In conclusion, the resistance allele(s) were quantitative and, thus, make selection for resistance difficult. Therefore, the introgression of the resistance allele(s) was not successfully completed. To determine the mechanism of resistance, the wild radish plants resistant WARR6-26 (R) and susceptible WARR7-5 (S) were treated with radiolabeled MCPA. There was no difference in metabolism of [14C]MCPA between R and S plants. Based upon the decline in the total 14C recovered over 72 h in R and S it was clear that both were “losing” [14C]MCPA; however, R plants were losing MCPA more rapidly. It was hypothesized that because R plants exude 14C more rapidly from their roots than S plants, this accounted for the resistance of R plants.

The complex vasculature seen in the vascular plants provides a scaffolding of structural support and is responsible for the movement of water, minerals, photosynthate and some hormones. The auxin canalization model proposes that a positive feedback mechanism causes auxin-transporting cells to become vascular cells. We have isolated a leaf-patterning mutant, unhinged (unh), which shows a simplified leaf vascular pattern with more freely ending veins and fewer secondary veins. Expression of the ATHB8::GUS reporter construct indicates that the UNH gene acts prior to procambial patterning of the first presumptive secondary veins. Expression of the auxin responsive reporter gene DR5::GUS is reduced in unh leaves and roots, indicating that UNH may be involved in auxin signaling. Increasing the level of auxin in unh leaves through the addition of auxin transport inhibitors, low concentrations of 2,4-dichlorophenoxyacetic acid, and through introducing unh into mutants in which auxin transport is defective partially rescues the unh phenotype, supporting this hypothesis. The unh mutation maps to a 60kb region near the top of chromosome IV. No other leaf vascular mutant or auxinassociated mutant have been reported in this area, thus UNH represents a novel component of leaf vascularization and auxin signaling. / xi, 65 leaves : ill. (some col.) ; 29 cm.

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Arabidopsis thaliana contains two genes annotated as methylthioadenosine nucleosidases (MTN): MTN1, At4g38800 and MTN2, At4g34840. This enzyme activity hydrolyzes the methylthioadenosine (MTA) produced by nicotianamine (NA), polyamine (PA), and ethylene biosynthesis to methylthioribose (MTR) within the Yang cycle. Comprehensive analysis of the mtn1-1mtn2-1 mutant line with 14 % residual MTN activity revealed a complex phenotype that includes male and female infertility and abnormal vascular development. Based on metabolite profiling, mtn1-1mtn2-1 has a reduced NA content, altered PA profiles with higher putrescine (Put) and lower spermidine (Spd) and spermine (Spm) levels, disrupted metal ion profiles, and abnormal auxin distribution. The modeling of Arabidopsis PA synthases developed by comparison with the crystal structures of human Spd and spermine synthases complexed with MTA suggests that Arabidopsis PA synthases are product inhibited by MTA. Thus, these pleiotropic mutant phenotypes possibly are the result of one metabolite directly inhibiting numerous pathways. By creating and analyzing a series of mutants and transgenic lines with moderate levels of MTN activity the complex phenotype of mtn1-1mtn2-1 was dissected in order to determine the fundamental trait associated with MTN deficiency. Two double mutants were identified by crossing single T-DNA mutants, and an artificial micro RNA (amiRNA) line was generated by transforming mtn1-1 with amiRNA specific to MTN2. The T-DNA double mutants, mtn1 4mtn2-1, and mtn1-1mtn2-5 had 98 % and 28 % MTN activity, respectively, whereas the amiRNA line has 16 % MTN activity. The growth, development, and metabolite analysis of these mutants revealed that their delayed bolting, correlated with an increased number of leaves, was the common trait observed across all lines. Xylem proliferation defects and increased number of vascular bundles per unit area were shared in all lines except mtn1 4mtn2-1. Based on these results, auxin distribution is proposed as the key target of the accumulated MTA that results from MTN deficiency. The infertility related to MTN-deficiency was restored by supplying 100 ??M of Spd to the mtn1-1mtn2-1 seedlings over 14 days. The data presented in this thesis reveals two potential links that work synergistically to recover fertility in this mtn1-1mtn2-1 line. Based on a detailed analysis of the female gynoecia morphology, transcript, hormone and metabolite profiles, it is proposed that the Spd partially reverses the mutant phenotypes through the recovery of auxin distribution and /or vascular development. Interestingly, the Spd effect seems to be transgenerational: they give rise to plants that are genotypically mtn1-1mtn2-1 but phenotypically WT over generations. Taken together, all of the results suggest that MTN-deficient mutants provide the potential for unraveling the molecular mechanism associated with nicotianamine, polyamines, auxin, and vascular development with respect to enhancing the efficiency of nutrient use and yields in plants.

Auxins are plant hormones involved in virtually all aspects of plant life. Despite long-term commercial and horticultural use of the auxin Indole-3-Butyric Acid (IBA), a full recognition of its natural occurrence in plants was made only recently. I have used multiple approaches to dissect the role of IBA in Arabidopsis thaliana. This thesis includes the first characterization of a mutant with an altered response to IBA that retains wild-type sensitivity to Indole-3-Acetic Acid (IAA), the most studied endogenous auxin. This mutant, named resistant to IBA ( rib1), has modified root architecture and gravitropism and is resistant to auxin transport inhibitors. As these phenotypes are reminiscent of those of characterized auxin transport mutants, movement of IAA and IBA was studied in wild-type and mutant plants. IBA is transported in seedlings in three distinct flows, like IAA, and this transport is saturable, indicating it is carrier mediated. However, unlike IAA, IBA is not polarly transported in inflorescence axes, and IBA transport is not sensitive to IAA transport inhibitors. These results suggest IAA and IBA transport could be mediated or regulated by different mechanisms. In rib1 seedlings, all flows of IBA transport are modified, while IAA transport levels are unchanged. Modifications in IBA transport match phenotypic differences between rib1 and wild-type, and analyses of the physiological effects of IBA also suggest IBA has a role in defining wild-type seedling morphology in Arabidopsis. Though IAA transport levels are not changed in rib1, one flow of IAA transport is rendered insensitive to IAA transport inhibitors, perhaps revealing cross-talk between IAA and IBA transport regulation. Additionally, double mutant analyses reveal that IAA transport and response mutants can suppress some phenotypes of rib1, and some mutant combinations produce novel phenotypes, further suggesting cross-talk between IBA and IAA transport and response p

Plants are stationary and need to adapt to the environment they live in. Integration of environmental cues, such as changes in light and temperature, can occur either directly or through the action of hormones. Hormone and light signaling leads to rapid changes in gene expression, and eventually changes in protein levels. In this thesis I have studied how the epigenetic regulator TERMINAL FLOWER2 (TFL2) is involved in light and hormonal signaling in the model organism Arabidopsis thaliana (thale cress). TFL2 is the only Arabidopsis homologue of HETEROCHROMATIN PROTEIN1 (HP1). HP1 proteins have been shown to be involved in repressing gene expression by maintaining the tight structure of heterochromatin or by forming a heterochromatin like structure in euchromatic regions. Unlike metazoan HP1 which can be localized both to eu- and heterochromatin, TFL2 is uniquely localized to euchromatin. tfl2 mutants have reduced levels of free auxin and a reduced rate of auxin biosynthesis. TFL2 binds to and promotes spatial and temporal expression of the genes belonging to the YUCCA gene family, which are believed to regulate a rate limiting step in the auxin biosynthesis pathway. Further, TFL2 binds to a subset of Aux/IAA proteins to repress auxin regulated genes involved in ovule and carpel development. In a similar way, TFL2 is also involved in repressing two jasmonate responsive genes, VEGETATIVE STORAGE PROTEIN1 and 2. This TFL2 regulated repression might occur through the interaction with the jasmonate responsive protein JAZ6. In light signaling TFL2 is involved in repressing both phytochrome A and B signaling as the response to red and far red light is enhanced in tfl2 mutants. The shade avoidance response and chloroplast biogenesis are also regulated by TFL2 as the hypocotyls of tfl2 are not able to elongate as wt in shade conditions and greening is delayed upon de-etiolation of tfl2 seedlings. This work shows that TFL2 has a repressive function in auxin, jasmonate and light signaling and for the first time we show that TFL2 is directly involved in promoting gene expression.

Development in multicellular organisms requires a strict balance between cell division and differentiation. The simple architecture of the Arabidopsis thaliana root makes it an ideal model for studying molecular mechanisms controlling both the transition from cell division to cell differentiation and cell fate determination. The class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factors (TFs) are well known developmental regulators, controlling important aspects of embryogenesis, shoot meristem activity, leaf polarity and vascular patterning. The HD-ZIP III TFs are under post-transcriptional control of microRNA165 (miR165) and miR166. In this thesis, I present a cell-to-cell signalling pathway underlying root vascular patterning and describe signaling pathways downstream of the HD-ZIP III TFs in their control of root development. The TF SHORTROOT (SHR), moves from the vascular stele cells to the surrounding endodermal cell layer. We show that SHR acts here to transcriptionally activate MIR165A and MIR166B, and the miR165/6 produced in the endodermis act non-cell autonomously to post-transcriptionally restrict HD-ZIP III mRNA levels in the peripheral stele. The resulting graded HD-ZIP III activity domain in the radial stele dose-dependently determines vascular cell type; high levels of HD-ZIP III in the central stele result in metaxylem formation while lower levels in the peripheral stele result in protoxylem. We provide evidence that the HD-ZIP III factors act as de novo xylem specifiers, because the quintuple mutant lacking all five HD-ZIP III genes forms no xylem. Furthermore, reducing the plasmodesmatal aperture through callose accumulation inhibits the bi-directional mobility of both signalling molecules, providing evidence that both SHR and miR165/6 move cell-to-cell via plasmodesmata to control root development. I present downstream components of the miR165/HD-ZIP III TFs in the root meristem, identified through a time-course induction of miR165 coupled to transcriptome analyses. This experiment revealed novel roles for HD-ZIP III TFs in vascular patterning and meristem size control. I show that HD-ZIP III directed repression of auxin hormone signalling in the xylem axis is essential for proper xylem differentiation. Furthermore, I provide data to show that they also control the balance of reactive oxygen species in the root meristem, thereby directing meristem size and ultimately controlling root growth.

Cocoa (Theobroma cacao L.) is an important cash crop in the coastal areas of Papua New Guinea (PNG). In 2000, a mutant cocoa genotype MJ 12-226, with abnormal growth characteristics was identified at the Cocoa and Coconut Institute of PNG. The mutant, found among progenies of the cross Scavina 12 x Nanay 149, was characterized by dwarfing, small and narrow leaves, a small root system and strong branching habit. This study was designed to improve the understanding of the dwarf cocoa mutant and identify its relevance to future cocoa farming. The specific objectives are to investigate the feasibility of using a dwarf mutant of cocoa as a commercial rootstock for various hybrid cocoa clones, to study the genetics of this dwarf mutant and the role of plant hormones in differentiating this mutant from the normal phenotype. This study was carried out at the Cocoa and Coconut Institute of PNG, Rabaul, Papua New Guinea, and at the University of Queensland, Gatton Campus, Australia. The comparative study of mutant and normal cocoa seedlings revealed distinct vegetative differences between the two genotypes at the nursery stage. The largest effects of the mutant genotype are on stem elongation, short internodes, multiple stems, root growth and leaf growth. The agronomic results indicated that the mutant rootstock significantly reduced tree vigour at least in the early years. When used as a rootstock, the mutant had a significant dwarfing effect on scion growth of cocoa clones in the nursery and persisted in the field 12 months after planting. After that, the cocoa clones grafted on mutant rootstock reverted to normal growth. It was postulated that the accumulation of endogenous growth substances and cambial activity between the scion (normal) and the rootstock overcame the dwarfing effect of the mutant genotype. Yield and vegetative data collection from this trial ceased on May 2006 and recommenced in May 2007 to allow trees to recover from heavy canopy pruning due to cocoa pod borer attack. This resulted in one year loss of yield and vegetative data from this experiment. The first two years’ yield data indicated no significant differences between mutant, normal segregants and commercial rootstocks. However, cocoa clones bud grafted on these mutant rootstocks produced significantly greater yield at higher planting density compared with lower densities and this was sustained for two years. However, it might be expected that competition among cocoa trees would increase with increased size of trees, favouring low density planting and reducing the differential performance of the trees under high density planting with time. The major advantages of reducing tree vigour are to improve the harvest index, ease of harvesting, pruning and overall management of the trees. The continuation of this study to collect mature tree yield data (from fifth to eighth years) would confirm these results. This work has also demonstrated for the first time that cocoa clones grafted on mutant rootstock can produce similar yields to clones grafted on normal or commercial rootstocks. Moreover, this study has also established that cocoa clones derived from orthotropic scions can yield the same as plagiotropic scions when grafted on to either mutant or normal rootstocks. Therefore, it is proposed that mutant rootstocks and orthtropic scions should also be considered for future use in cocoa farming. The strong branching habit and dwarfing stature of the mutant suggested that the mutation affects the quantity or the balance of plant growth hormones, or both. The effect of exogenously applied gibberellic acid (GA3) on developmental processes in dwarf mutant and normal cocoa seedlings was studied. Both dwarf mutant and normal one month old cocoa seedlings responded to gibberellic acid (GA3) by accelerated longitudinal growth, especially seedling height. Paclobutrazol (PBZ) applied to normal cocoa seedlings altered the growth so that they almost resembled the mutant phenotype. It was postulated that PBZ prevented GA biosynthesis in the normal cocoa seedlings and therefore decreased bioactive auxin (IAA) which, in turn, then promoted lateral shoot growth; PBZ also slightly retarded plant growth. Synthetic auxin (IBA/NAA) significantly inhibited lateral bud sprouting and growth of intact, decapitated and nodal segments of mutant and normal cocoa seedlings compared with control and those treated with IAA. It appears that the mutant and normal cocoa genotypes have a similar capacity to metabolize IAA and this capacity was much greater than for synthetic auxins. The results suggested that auxin levels in the mutant seedlings were probably limiting, and therefore affected biosynthesis of gibberellic acid thus resulting in the dwarfing characteristics of the mutant cocoa genotype. However, analysis of endogenous IAA and GA1 levels at four months old did not reveal differences between the mutant and normal seedlings. The present genetic studies which included backcrossing, test crossing and selfing of F1 progenies confirmed earlier work on the mutant and showed that the mutant was probably heterozygous (Dd). The allele (DD) in mutant cocoa is probably lethal in nature because the phenotype of the homozygote was not observed amongst the mutant progenies. Mutant segregants were obtained in a 1:1 ratio only when the mutant clone was used as the female; however, when the mutant was used as male, very few mutant segregants were obtained. This suggested selective pollen viability (pollen grains carrying the D allele are nonviable) or an incompatible reaction between pollen tubes (D carrying pollen) and style or ovule. The selfing and test-crosses of F1 progenies resulted in deviation from the expected 3:1 segregation ratio to a 1:1 ratio.

This thesis describes a molecular-based study undertaken to analyse the expression of the RAMOSUS1 (RMS1) and RAMOSUS5 (RMS5) genes in pea (Pisum sativum). Both genes encode carotenoid cleavage dioxygenase (CCD) enzymes that are together proposed to control the synthesis of an inhibitor of bud outgrowth termed SMS (Shoot Multiplication Signal). SMS was recently identified as strigolactone. Expression analyses of RMS1 presented here have built upon earlier experiments which demonstrate it to be a highly regulated transcript. RMS1 mRNA levels are known to be rapidly decreased following removal of the shoot apex but are subsequently restored to that of intact plants by auxin (indole-3-acetic acid or IAA). This regulatory mechanism is retained in all five ramosus mutants tested to date. Together with physiological data, this indicates RMS1, and therefore SMS, are required in IAA-mediated suppression of bud outgrowth. Another significant aspect of RMS1 regulation identified in previous studies involves a graft-transmissible, long-distance feedback signal that moves from shoot to root. This feedback regulation is dependent on the RMS2 gene and enhances RMS1 expression levels. Prior to the cloning of RMS5 and its discovery as a second CCD enzyme in the RMS network, reciprocal grafting studies with the rms mutants indicated RMS5 may act in the same pathway as RMS1 to produce SMS. Multiple studies presented here demonstrate that these two CCD genes are expressed in similar tissues and are regulated by the same signals, specifically IAA and the RMS2-dependent feedback signal. Like RMS1, the RMS5 gene also retains its IAA response in the rms mutants. However, RMS5 is generally less responsive to changes in IAA and RMS2-dependent feedback, as it exhibits smaller fluctuations than RMS1 in its expression levels. Together these findings support a general view that RMS1 is more likely to control a rate-limiting step in SMS synthesis. A previous study indicated that RMS1 expression may be up-regulated by IAA through a posttranscriptional mechanism. This thesis sought to more closely examine the RMS1 and RMS5 IAA response by separately observing the effect of IAA on subsequent transcription. New transcripts, termed heterogenous nuclear RNAs (hnRNAs), were relatively quantified in parallel with existing mRNAs in the steady-state cytoplasmic pool. The experiments conducted here provide further evidence that IAA may act post-transcriptionally to stabilise RMS1 mRNA because the changes in hnRNA are not proportional to the changes in mRNA following IAA-modifying treatments. IAA may still function to induce transcription of RMS1, but this does not appear to be a significant mechanism by which IAA regulates RMS1 expression. In contrast, the IAA induction of RMS5 occurs predominantly via new transcription and RMS5 either lacks or is not as strongly subjected to the IAA-mediated mRNA stabilisation mechanism proposed for RMS1. Initial studies described in this thesis also suggest that IAA could act to regulate the expression of the Arabidopsis orthologues MORE AXILLARY BRANCHING (MAX) genes via a post-transcriptional mechanism. Analyses of MAX hnRNA and mRNA levels in Arabidopsis to date indicate it is the RMS5 orthologue MAX3 which exhibits an IAA response most like RMS1. Additional studies into the regulation of RMS1 and RMS5 presented in this thesis provide further insights into the molecular mechanisms controlling their expression levels. In vitro experiments with the translation inhibitor cycloheximide demonstrate that RMS5 expression levels are increased when protein synthesis is reduced, as previously shown for RMS1. Relative quantification of RMS1 and RMS5 hnRNA levels further demonstrate that the induction by cycloheximide is due primarily to an increase in new transcription, indicating that RMS1 and RMS5 are negatively regulated by a rapidly turned-over transcriptional repressor. Tissue specific effects on RMS1 expression were also observed which are consistent with a protein degradation function of the RMS4 F-box in the shoot. This thesis provides further evidence to suggest that SMS acts in concert with IAA to inhibit the sustained outgrowth of axillary buds. RMS1 and RMS5 expression levels are not regulated by a hypothetical fast decapitation signal which is proposed to cause the initial bud outgrowth occurring prior to decapitation-induced IAA depletion. RMS1, RMS5 and SMS are therefore unlikely to control the initial exit of buds from dormancy to an intermediate transition state. Studies here also suggest that enhanced shoot auxin transport and cytokinin biosynthesis are associated with axillary bud outgrowth because the rms mutants contain elevated shoot expression levels of a gene encoding the auxin efflux carrier PIN1 and two genes controlling cytokinin biosynthesis. Several approaches described in this study were used to characterise the RMS1 and RMS5 proteins. Anti-peptide antibodies were generated against both proteins and the results obtained show that although the antibodies are likely to recognise the full-length proteins, further work is required to effectively detect RMS1 and RMS5 in plant tissues via western blotting. Preliminary in situ immunolocalisation results indicate the RMS1 and RMS5 proteins are localised to the vasculature, consistent with gene expression analyses.