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    Ankyrin2 is essential for neuronal morphogenesis and long-term courtship memory in Drosophila.
    (BioMed Central Ltd, 2023-05-16) Schwartz S; Wilson SJ; Hale TK; Fitzsimons HL
    Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories.
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    Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis
    (BioMed Central Ltd, 2024-12) Tan WJ; Hawley HR; Wilson SJ; Fitzsimons HL
    BACKGROUND: Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14-3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. RESULTS: Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. CONCLUSIONS: We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.
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    A molecular analysis of flowering in Metrosideros : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Jury, Alexa
    A calendar of floral and vegetative developmental events has been previously determined for the woody perennial Metrosideros excelsa. M. excelsa flowers just once a year in summer and bud development has been related to seasonality and gene expression. M. collina cv. Tahiti is closely related to M. excelsa but appeared to flower sporadically through out the year. Using histology, the bud development of M. collina has been analysed and a developmental sequence determined. Periodicity of bud development has also been observed by obtaining bud measurements. While it is difficult to relate bud size to developmental phase or determine whether the bud is vegetative or floral, it appears that M. collina goes through two periods of bud development in one year and that flowering may be related to warm temperature. The genetic interactions between pathways controlling floral meristem development have been analysed in the herbaceous annual model plant Arabidopsis thaliana. In Arabidopsis, expression of the floral meristem identity gene, LEAFY (LFY), is regulated directly by the florally promotive gibberellin and photoperiodic pathways. LFY expression in Arabidopsis was upregulated in response to gibberellin application, which hastened the transition from vegetative to floral meristem. Another floral meristem identity gene, APETALA1 (AP1) is upregulated by LFY to promote formation of the floral meristem. However, exogenous application of gibberellic acid (GA3 ) to woody perennial trees, including M. collina, inhibits flowering. Due to the differences between Arabidopsis and woody perennial species, it is important to determine if the Arabidopsis model is a useful tool to analyse flowering in Metrosideros species. LFY and AP1 homologues were isolated previously from M. excelsa, and from M. collina in this project. The responsiveness of MEL (M. excelsa LFY-like) and MESAPI (M. excelsa AP1-like) and MCL (M. collina LFY-like) and MTAP1 (M. collina AP1-like) to GA3 , and other growth regulators and hormones was examined in juvenile and adult Metrosideros. MEL or MCL expression was not detected in juvenile or adult GA3 -treated Metrosideros, which supports GA3 inhibition of flowering in perennials. It appears that the gibberellin component of the Arabidopsis model does not represent appropriately, the response of perennial woody species to gibberellins in terms of flowering.
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    Molecular genetic analysis of plant Mei2-like genes : 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, 2002) Alvarez, Nena de Guzman
    Molecular and genetic methods were used to analyse how a novel class of genes, plant Mei2-like genes may be involved in the regulation of morphogenesis in plants. The study specifically aimed to 1) further characterise maize te1 (the first plant Mei2-like gene to be genetically analysed) and understand the morphological basis of the te1 mutant phenotype and 2) analyse the function of Arabidopsis Terminal Ear Like (TEL) genes using expression analyses and reverse genetics strategy. te1 maize mutants are initially characterised by abnormal phytomer formation and development. A more detailed morphological analysis shows that mutant plants 1) have smaller vegetative shoot apices than the wild type, 2) initiate leaves at a higher, more distal position on the apical dome and 3) have higher plastochron ratio. Molecular analyses of kn1 expression pattern, a marker of leaf founder identity, show that dowregulation of kn1 transcripts occur higher up the dome. Clonal analyses show that fewer number of leaf founder cells are recruited to form the leaf. TEL1 and TEL2 are expressed in distinct overlapping domains in the undifferentiated region of the shoot apical meristems during the embryo, vegetative and reproductive stages of Arabidopsis development suggesting involvement of these genes in regulating meristem development and subsequent maintenance. The distinct expression of TEL1 in both the embryonic SAM and RAM raises the possibility of a unifying regulatory mechanism in the formation of the root and the shoot. The absence of TEL single knockout phenotypes supports the idea of functional redundancy. When the TEL genes were both knocked out, double mutant phenotypes show apical-basal pattern defects, ectopic production of numerous secondary shoots, production of numerous leaves and basic embryonic pattern defects such as deletions of apical and/or basal region of the seedling. Results of this study support the hypothesis that plant Mei2-like genes are important in regulating morphogenesis in plants and that they are required in the early patterning of the basic plant body.
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    Characterisation of tomato MADS-box genes involved in flower and fruit development : 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, 2001) Ampomah-Dwamena, Charles
    MADS-box genes encode transcription factors that are involved in various aspects of plant development, by regulating target genes that control morphogenesis. Over the last decade, plant MADS-box genes have been studied extensively to reveal their control of floral development, especially in the model plants Arabidopsis and Antirrhinum. Their functions are however, not restricted to the flower but are involved in various aspects of plant development (Rounsley et al., 1995; Jack, 2001). By virtue of their extensive roles in the flower, these genes are expected to function in fruit development, which is a progression from flower morphogenesis. The aim of this study was to examine the role of MADS-box genes during flower and fruit development. Two new members of the tomato MADS-box gene family, TM10 and TM29 were identified. TM29 was isolated from a young fruit cDNA library by screening with homologous MADS-box fragments and TM10 was amplified by polymerase chain reaction from fruit cDNA templates. These genes were characterised by sequence and RNA expression patterns and their functions examined using molecular genetic techniques. Sequence analyses confirmed that both genes belong to the MADS-box family. TM29 shows 68% amino acid sequence identity to Arabidopsis SEP1 MADS-box protein. TM29 expression pattern showed similarities as well as differences to SEP1 (Flanagan and Ma. 1994). TM29 is expressed in shoot, inflorescence and floral meristems unlike SEP1, which is expressed exclusively in floral meristems (Flanagan and Ma. 1994). TM29 is expressed in all the four whorls of the flower. During floral organ development, it is highly expressed at early stages of the organ primordium but decreases as the organ differentiates and matures. In the mature flower bud, TM29 is expressed in the anther and ovary pericarp. During fruit development, TM29 is expressed from anthesis ovary to fruit of 14 days post-anthesis with its transcript localised to the pericarp and placenta. TM10 showed 64% amino acid identity to Arabidopsis AGL12. across the entire sequence. This notwithstanding, TM10 expression differed from AGL12. TM10 was expressed in shoot tissues of tomato and was not detected in roots. In contrast, the AGL12 gene transcript was only present in the roots of Arabidopsis (Rounsley et al., 1995). Expression was detected in leaves, shoot growing tips, floral buds and fruit. During fruit development, TM10 is expressed in anthesis ovary and in fruits at different growth stages. The functions of TM29 and TM10 were examined by transgenic techniques and phenotypes generated were consistent with their spatial and temporal gene expression patterns. TM29 transgenic phenotypes suggested it might be involved in the control of sympodial growth, transition to flowering, proper development of floral organs. parthenocarpic fruit development and maintenance of floral meristem identity. TM10 affected apical dominance and flowering time, development of floral organs and parthenocarpic fruit development.
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    Leaf morphogenesis and tillering behaviour in single plants and simulated swards of Guinea grass (Panicum maximum Jacq.) cultivars : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Natural Resources, Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Carvalho, Dora Duarte de
    In a series of five experiments carried out in growth cabinet and glasshouse environment, potted plants of Guinea grass (Panicum maximum Jacq.) cultivars were subjected to different cutting heights and environmental conditions. Herbage accumulation, leaf and tiller morphogenesis, leaf tissue turnover and tiller dynamics were evaluated. In one study three cultivars, Aruana, Mombaça and Tanzânia were grown for 18 months in miniature swards where plants were subjected to two defoliation heights (150 and 300mm), and productivity and tiller dynamics were evaluated. Cultivar and defoliation intensity effects on herbage production, leaf morphogenesis and tiller population density were observed. Herbage DM harvested from Mombaça was 32.3 and 31.7% more than Tanzânia and Aruana, respectively. There were also seasonal effects on tiller appearance rate and tiller death rate associated with flowering. A greater proportion of tillers flowered in cv. Tanzânia than cv. Mombaça. Two further experiments quantitatively described leaf turnover and associated morphogenetic characters in potted plants of the same three cultivars. One of these carried out in a growth cabinet included three defoliation regimes (50 mm, 200 mm, or uncut) and leaf turnover and the distribution of various tiller categories within the plants were measured. The other, carried out in a glass house, examined leaf turnover and site filling responses in undefoliated established plants. Higher temperature in the growth cabinet increased LAR, LER, FLL, and senescence and reduced site filling, LLS and NLL, and these effects were greater than cultivar effects. Severe defoliation (50 mm) initially resulted in increased tiller number per plant in all cultivars, but later decreased tiller number per plant through tiller death. The next experiment was established to determine limits of plasticity in potted plants of cv. Mombaça and cv. defoliation intensities (100, 200 and 400 mm height). Herbage growth and net accumulation declined progressively, and senescence increased with increasing defoliation height in both cultivars. Treatment effects on root mass and root development were similar to those on herbage production. There was evidence of serious decline in vigour of aging tillers in both cultivars, and of substantial differences in the contribution of individual tiller cohorts to production, independent of the age effect. A final experiment using 14C showed that translocation of assimilates from primary tillers to daughter and secondary tillers was greater in cv. Tanzânia than cv. Mombaça. The implications of these studies to understanding of the morphogenetics and production potential of these cultivars, their relevance to management decisions, and requirements for further studies are discussed.
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    Morphological, physiological, and molecular studies on the effect of shoot architecture on phase change and floral transition in Eucalyptus occidentalis and Metrosideros excelsa : 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, 2007) Jaya, Elizabeth S.K.D.
    Shoot morphogenesis in Eucalyptus occidentalis and Metrosideros excelsa was analysed at the morphological, physiological and molecular levels to understand the regulation of phase change and the floral transition. Study of the regulation of these developmental plant processes is limited in woody species due to their long juvenile phase. Six ecotypes of E. occidentalis were grown to two predetermined architectures (free branching or single stem). Free branching plants of ecotype 13648 displayed adult shoot phenology (lanceolate leaves) earlier than single stem counterparts. In addition, changes in leaf morphology in free branching plants were accompanied with changes in leaf anatomy and gas exchange signifying that in E. occidentalis complexity of shoot architecture had a significant effect on rate of phase change. Flowering was observed in all but one ecotype irrespective of architecture demonstrating that vegetative phase change and floral transition are temporally uncoupled in this species. To understand the floral transition at the molecular level in E. occidentalis, partial homologues of the inflorescence meristem identity gene TERMINAL FLOWER1 and floral meristem identity genes LEAFY and APETALA1 were isolated. The expression patterns of these meristem identity genes during development of free branching and single stem plants were analysed by quantitative real-time PCR. Increased levels of expression of EOLFY and EOAP 1 (relative to α -TUBULIN) were displayed at more proximal nodes in free branching plants than in single stem plants. Elevated floral meristem identity gene expression levels correlated with flower initiation. Further, effects of architecture and environment on gene expression were monitored in E. occidentalis. The overriding effect of shoot architecture on the floral transition was observed under warm long day and ambient environments. Elevated levels of EOLFY and EOAP 1 were correlated with floral bud score and EOAP1 was found to be a reliable marker of floral transition in E. occidentalis. Low levels of EOTFLI expression were detected in buds irrespective of their position on the plant leading to the suggestion that this might have contributed to the precocious flowering observed in this species. In contrast to E. occidentalis, M excelsa attained adult shoot phenology (pubescent leaves) faster when grown as single stem plants than as free branching plants. It appears that growth as height is required for vegetative phase change in this species. However, floral transition occurred only once single stem plants were allowed to branch. Vegetative phase change and the transition to flowering seem to be coordinated in this species with the former being a pre-requisite for the latter.