Thesis (M. Sc.(Agric.))(Agronomy)--University of Pretoria, 2008. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

This thesis presents an investigation into the development of plasmodesmata (PD), which are specialised pores in plant cell walls through which the cytosol and membranes of neighbouring cells are linked. Modification of PD from their initial single-tube (‘simple’) structures to branched (‘complex’) structures is an important part of tissue maturation as it allows cells to restrict the movement of syplasmically mobile molecules including hormones, RNAs and proteins. Conversion of PD from simple to complex is co-ordinated across large populations of cells to produce symplasmic domains, transport barriers, and preferential transport pathways. The development of PD is therefore intrinsic to the wider development and morphogenesis of cells, tissues, and organs. The aim of this project was to investigate the development of PD from simple to complex, particularly during the predictable, large-scale conversion of PD structure that accompanies the leaf transition from sink state to source. To study this I used transgenic plants expressing a GFP-tagged viral protein which accumulates specifically in complex PD, while leaving simple PD unlabelled. The project follows the development of complex PD from the early stages of leaf development to maturity using a range of microscopy techniques. Structured illumination microscopy was used to view labelled PD at super resolution, which gave new structural details about complex PD using a breakthrough technology. Conventional and high-throughput confocal and electron microscopy were used to localise PD within tissues in a broad survey of PD location in leaves to identify patterns of PD development. An imaging chamber was developed that allowed the development of complex PD to be viewed in real time and identified conditions that can trigger structural conversion of PD. Finally, a high-throughput microscopy study was performed to identify how hormones, sugar availability, environmental stresses, defence responses and inhibitors can affect PD development.

Les plasmodesmes sont des nanopores membranaires qui traversent la paroi des cellules végétales. Ces nanostructures jouent un rôle central en communication intercellulaire et agissent comme des centres de signalisation capables de générer et de relayer le signal de cellule à cellule via l’activité de récepteurs. En tant qu’éléments clés de la communication intercellulaire, les plasmodesmes coordonnent les processus liés à la croissance et au développement des plantes ainsi que les réponses aux stress environnementaux. Dans cette étude, nous avons identifié trois récepteurs de la famille des récepteurs riches en leucine (LRRRLK), capables de relocaliser de manière dynamique au niveau des plasmodesmes après un stress abiotique. L'association plasmodesmale est rapide et survient en moins de 2 minutes. Cette association dynamique n’est pas un comportement général des protéines associées à la membrane plasmique ou aux microdomaines membranaires. En focalisant notre étude sur SAK1 (Sucrose Activated Kinase) nous avons démontré que l'association dynamique aux plasmodesmes est indépendante de la composition en stérols ou en sphingolipides et est partiellement dépendante du statut de phosphorylation de cette protéine. Nous avons identifié un strech d'acide aminé polybasique dans le domaine Juxtamembrane (JMD) de SAK1, décrit chez l’homme comme interagissant avec les lipides anioniques, qui est indispensable à l'association conditionnelle aux plasmodesmes. Au total, nos données indiquent que les changements dans la membrane signature moléculaire des domaines spécialisés plasmodesmes accompagne les réponses aux stimuli externes. / Plasmodesmata pores sustain intercellular communication in plants. They act as specialized signalling hubs clustering receptor activities, and are capable of generating and relaying signalling from cell-to-cell. As key elements in intercellular communication, plasmodesmata coordinate processes related to plant growth, development and environmental stresses responses. In this study, we identified three PM-located Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs) that are able to dynamically and conditionally relocate to plasmodesmata upon abiotic stress. Plasmodesmal association occurs within 2 minutes and is not a general behaviour of PM or microdomain-associated proteins. Focusing on SAK1 (SUCROSE ACTIVATED KINASE) we demonstrated that the plasmodesmal dynamic association is neither dependent of sterol or sphingolipid composition and nor driven by the protein phosphorylation status. Importantly, we identified a polybasic amino acid motif in the Juxtamembrane Domain (JMD) of SAK1 predicted to bind anionic lipids that is critical for conditional plasmodesmal association. Altogether our data indicate that changes in the membrane molecular signature of plasmodesmata specialized domains accompanies responses to external stimuli.

Sterols have been identified as major components of membrane lipids that are part of specialized membrane domains necessary for organizing events such as polar protein targeting and signal transduction in plants, fungi and animals. However a common modification of sterols is the addition of sugar moieties via glycosylation abundantly found in plants. An exact physiological role for such diversification of sterols in plants is still unknown. Using reverse genetics and transcriptomics we show that UDP-glucose: sterol glucosyltransferase encoded by UGT80B1 is necessary for correct epidermal patterning in Arabidopsis root. Patterning of hair cells (trichoblasts) and non-hair cells (atrichoblasts) in the epidermis of the Arabidopsis root involves signaling through SCRAMBLED (SCM), a plasma membrane localized LRR-RL kinase. Feedback regulation via the transcriptional regulatory complex containing R2R3-MYB transcription factor WEREWOLF (WER) represses SCM and activates the homeodomain-leucine-zipper protein GLABRA2 (GL2) in atrichoblasts. Evidence suggests symplastic connections between cells, known as plasmodesmata, establish passage ways for single-repeat R3-MYB transcription factors to activate SCM expression in trichoblasts. Mutations in UGT80B1 cause atypical localization patterns of GL2, WER, and SCM in the root epidermis. The ugt80B1 formed fewer trichoblasts in comparison to wild-type. A translational fusion of UGT80B1 to GFP localizes to the ER, plasma membrane and to sites that appear to be plasmodesmata-associated desmotubules. Ultrastructural analysis revealed abnormalities in plasmodesmata formation and morphology in ugt80B1 mutants. Steryl glucoside profiling indicated deficiencies in specific glycosylated sterol compounds in roots. This study identifies UGT80B1 as a novel membrane component that is critical for plasmodesmata morphogenesis and cell-fate determination in the root epidermis. A model is proposed in which UGT80B1 activity provides spatially discreet sterol and steryl glucoside architecture within the plasma membrane to anchor the SCM receptor and within plasmosdesmata to facilitate intercellular movement of R3-MYB regulatory proteins underlying proper differentiation of trichoblasts versus atrichoblasts. Moreover, evidence from reverse genetics, proteomics and live cell imaging point to a actin dependent localization of UGT80B1 at the vesicle rich zone of root hair tip. This localization actively supports root hair elongation via tip growth, possibly by membrane modifications required for vesicle trafficking.

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.

Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Jung-Youn Lee, Dept. of Plant & Soil Science. Includes bibliographical references.

This study focused on the primary cell wall constituents and plasmodesmata (PD) density in three mosses and four liverwort apical cells (AC) and immediate derivatives. The three mosses have tetrahedral apical cells and the liverworts possess tetrahedral, hemidiscoid and lenticular AC geometries. The primary cell wall in the studied taxa is comprised of two layers. A fibrillar layer, which is the outer wall layer, contains compacted cellulosic fibrils, and represents the two adjacent primary walls and middle lamella, the latter of which is rarely discernible. An electron-lucent inner wall layer abuts the plasma membrane. This layer has faint fibrous materials that extend from the plasma membrane to the fibrillar layer. Generally, as the cell wall ages it thickens, the fibrillar layer increases in width while the electron-lucent wall stays more or less consistent in width. In the four liverworts, the most recent wall of the AC has the highest PD density in the apical region regardless of AC geometry. As the walls elongate, primary wall is laid down between PD, separating them and resulting in lower densities and wider PD diameters in older walls. The season of fixation and whether plants were studied from nature or culture have an influence on AC ultrastructure. A developmental study of Physcomitrella patents gametophores in four stages, bud, 2-leaved, 7-8-leaved and ~20-leaved, reveals that the primary cell wall constituents change slightly during development. Specifically, LM5 a RG-I pectin antibody against the galactan branch epitope is only localized in the fibrillar layer of young water-conducting cells in the 7-8-leaved and 20-leaved gametophores. LM20, an antibody against HG esterified pectins, does not localize in any of the cell walls during development. The distribution patterns for AGPs (JIM13 and LM2) are consistent during gametophore development and predominantly localize on the electron-lucent layer and wall/plasma membrane interface. However, LM2 is mainly localized on the fibrillar layer in 7-8-leaved cell walls. AGPs also localize on element of the cytoplasm. LM6, an antibody against an RG-I pectin with arabinan branch epitopes, also localizes AGPs and because it expressed similar distribution patterns as JIM13 and LM2 on the cell wall, it likely localizes AGP in Physcomitrella. In addition, LM6 localizes pectins on the fibrillar layer similar to LM5 and LM19 for HG unesterified pectins. Callose predominantly localizes at the PD neck region. This study provides the first documentation of changes in size and shape of AC with age in Physcomitrella patens gametophores. The PD densities of gametophytes examined in this study fall into the lineage-specific network of PD (LPD) group designated for sporophytes of monilophytes and Selaginella (heterosporous lycophyte) with single ACs. Takakia lepidozioides leafy shoot has a tetrahedral AC with a highly curved free surface. This peculiar moss has mucilage hair (MH) associated with axil of phyllids. Mucilage hair in both species are 3-celled with a forth epidermal cell as the base. However, occasional 2-celled MH is seen in T. ceratophylla. The ultrastructure of MH has similarities with other mosses and liverworts.

Les Rémorines (REM) du groupe 1 sont des protéines spécifiques du monde végétale. Malgré leur caractère hydrophile elles sont localisées à la membrane plasmique. La phosphorylation des REM serait potentiellement impliquée dans la signalisation précoce et la défense des végétaux contre les pathogènes. Benschop et al. (2007) détecte AtREM1.3 (Arabidopsis thaliana, groupe 1b) phosphorylée en réponse au traitement par l'éliciteur générale flg22, tandis que Widjaja et al. (2008) a suggéré que la phosphorylation de AtREM1.2 est potentiellement impliquée dans la signalisation précoce à l'infection par Pseudomonas syringae. La fonction précise de la phosphorylation des protéines REM du groupe 1 reste inconnue. Des travaux antérieurs dans le laboratoire ont montré que le mouvement du virus X de la pomme de terre (PVX) est inversement corrélée à l'accumulation de StREM1.3 (Solanum tuberosum) et que StREM1.3 peut interagir physiquement avec la protéine de mouvement TRIPLE GENE BLOC Protein 1 (TGBp1) du PVX (Raffaele et al., 2009). Dans ce travail, nous avons étudié les mécanismes qui sous-tendent les interactions REM-TGBp1 et nous avons essayé de caractériser biochimiquement la kinase qui phosphoryle REM. Les conséquences physiologiques de l'interaction TGBp1 / StREM1.3 et de la phosphorylation de REM en terme de propagation des virus, d’inactivation génique post-transcriptionnelle, de régulation de l’ouverture des plasmodesmes, et d’activation de kinase ont également été étudiés. / The group 1 Remorin (REM) proteins are plant-specific oligomeric proteins that have been reported to localize to the plasma membrane despite their overall hydrophilic nature. There is evidence that the REM protein phosphorylation is potentially implicated in the early signaling and defense. Benschop et al. (2007) detected the AtREM1.3 (Arabidopsis thaliana group 1b of REM protein family) to be phosphorylated in response to treatment with the general elicitor flg22, while the Widjaja et al. (2008) suggested that the phosphorylation of AtREM1.2 is potentially implicated in early signaling upon infection with Pseudomonas syringae. The precise exact function of the group 1 REM protein phosphorylation remains unknown. Previous work in the laboratory showed that Potato virus X (PVX) movement is inversely correlated to potato StREM1.3 accumulation and that StREM1.3 can physically interacts with the movement protein TRIPLE GENE BLOCK PROTEIN 1 (TGBp1) from PVX (Raffaele et al., 2009). In this work, we studied the mechanism underlying the REM-TGBp1 interactions and we tried to characterise biochemically the kinase that phosphorylate REM. The physiological consequences of TGBp1/ StREM1.3 interaction and REM phosphorylation in terms of virus spreading, post-transcriptional gene silencing, plasmodesmata gating, kinase activation were also investigated.

abstract: The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants. / Dissertation/Thesis / 3D MODEL OF SIMPLE PLASMODESMATA / 3D MODEL OF COMPLEX PLASMODESMATA / MODELING SIMPLE PLASMODESMATA IN IMOD / MODELING COMPLEX PLASMODESMATA IN IMOD / Doctoral Dissertation Biology 2016

The effect of cytokinin and gibberellin, and in particular a combination of the two, on termination of dormant potato tubers was investigated. The objective was to effectively terminate dormancy through the external application of a combination of cytokinin and gibberellin. Freshly harvested tubers were treated and either cut at the stolon end with the apical portions placed on moist cotton wool, or left intact and dry. Tuber segments treated with a high concentration of cytokinin (0.1g.Lˉ¹) or a combination of cytokinin and gibberellin sprouted within 5 days, whereas high gibberellin concentrations (0.1g.Lˉ¹) stimulated sprouting within 9 days. Untreated tuber segments supplied only with moisture terminated dormancy later than hormonal treated tubers, but much earlier than segments that were kept dry. Tuber segments treated with a combination of cytokinin and gibberellin, or a high concentration of gibberellin (0.1g.Lˉ¹), produced more and longer sprouts than tubers treated with only cytokinin (0.1g.Lˉ¹) or a low concentration of gibberellin (0.005g.Lˉ¹). Sprouts on tuber segments treated with a combination of cytokinin and gibberellin attained maximum sprout growth rate nine days after treatment, but thereafter the growth rate decreased. This decrease may be a consequence of closed plasmodesmata although membrane permeability and its affect on assimilate availability may play a role. This phenomenon deserves further research attention. Removal of wound periderm did not reactivate sprout growth. The wounding of tubers by removing a portion at the stolon end and supplying moisture greatly enhanced the termination of dormancy and subsequent sprout growth, indicating that the availability of water may be a factor in initiation of sprouts. The results are compatible with the hypothesis that cells in dormant buds are arrested in the G1 phase of the cell cycle. Cytokinin is needed to initiate cell cycling, but gibberellin is also needed to initiate and maintain cell growth. These two growth regulators are also involved in the opening of the plasmodesmata as well as the creation of new plasmodesmata witch would establish communication between the apical meristem and the rest of the tuber. / Dissertation (MSc(Agric))--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted