https://ogma.newcastle.edu.au/vital/access/ /manager/Index ${session.getAttribute("locale")} 5 https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:36482 Wed 20 May 2020 16:19:05 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:26914 cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca²⁺ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca²⁺ signal intensity, by withdrawing extracellular Ca²⁺ or blocking Ca²⁺ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca²⁺ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca²⁺ by co-operative functioning of plasma membrane Ca²⁺-permeable channels and Ca²⁺-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca²⁺ signal was organized into discrete patches that aligned spatially with clusters of Ca²⁺-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca²⁺ were consistent with inward-directed plumes of elevated [Ca²⁺]_cyt. Plume formation depended upon an alternating distribution of Ca²⁺-permeable channels and Ca²⁺-ATPase clusters. On further inward diffusion, the Ca²⁺ plumes coalesced into a uniform Ca²⁺ signal. Blocking or dispersing the Ca²⁺ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca²⁺ plumes define the loci at which wall ingrowth papillae are deposited.]]> Wed 11 Apr 2018 16:31:50 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:28150 Vicia faba (L.) is an important cool-season grain legume species used widely in agriculture but also in plant physiology research, particularly as an experimental model to study transfer cell (TC) development. TCs are specialized nutrient transport cells in plants, characterized by invaginated wall ingrowths with amplified plasma membrane surface area enriched with transporter proteins that facilitate nutrient transfer. Many TCs are formed by trans-differentiation from differentiated cells at apoplasmic/symplasmic boundaries in nutrient transport. Adaxial epidermal cells of isolated cotyledons can be induced to form functional TCs, thus providing a valuable experimental system to investigate genetic regulation of TC trans-differentiation. The genome of V. faba is exceedingly large (ca. 13 Gb), however, and limited genomic information is available for this species. To provide a resource for future transcript profiling of epidermal TC differentiation, we have undertaken de novo assembly of a genome-wide transcriptome map for V faba. Illumina paired-end sequencing of total RNA pooled from different tissues and different stages, including isolated cotyledons induced to form epidermal TCs, generated 69.5 M reads, of which 65.8 M were used for assembly following trimming and quality control. Assembly using a De-Bruijn graph-based approach generated 21,297 contigs, of which 80.6% were successfully annotated against GO terms. The assembly was validated against known V faba cDNAs held in GenBank, including transcripts previously identified as being specifically expressed in epidermal cells across TC trans-differentiation. This genome-wide transcriptome map therefore provides a valuable tool for future transcript profiling of epidermal TC trans-differentiation, and also enriches the genetic resources available for this important legume crop species.]]> Wed 11 Apr 2018 16:06:55 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:14467 Wed 11 Apr 2018 14:35:38 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:20020 Wed 11 Apr 2018 14:02:44 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:26903 Vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. All functional groups of genes examined were expressed before and following transition to a transfer cell fate. What changed were the isoform profiles of expressed genes within functional groups. Genes encoding ethylene and Ca²⁺ signal generation and transduction pathways were enriched during uniform wall construction. Auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and ABA genes were evenly expressed across ingrowth wall construction. Expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. Uniform wall and wall ingrowth specific expression of exocyst complex components and SNAREs suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. Key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. Transfer cell specific expression of cellulose synthases was absent. Rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. More striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. Extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. Ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of ABC transporters. Sugar transporters were less represented. Conclusions: Pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. Each phase of ingrowth wall construction was linked with unique cohorts of expressed genes.]]> Wed 11 Apr 2018 13:16:39 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:26902 2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed.]]> Wed 11 Apr 2018 12:43:42 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:27813 Wed 11 Apr 2018 12:10:22 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:24956 Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal-enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal-enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons.]]> Wed 11 Apr 2018 10:50:02 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:47611 Vicia faba cotyledons. When these cotyledons are placed in culture, their adaxial epidermal cells trans-differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (_apoH₂O₂), and cytosolic Ca²⁺ ([Ca²⁺]_cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans-differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular _apoH₂O₂ directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca²⁺]_cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly.]]> Tue 24 Jan 2023 11:28:41 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:1319 Sat 24 Mar 2018 08:32:32 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:1437 Sat 24 Mar 2018 08:28:14 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:21810 trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.]]> Sat 24 Mar 2018 07:59:23 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:26905 Sat 24 Mar 2018 07:23:35 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:32294 Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.]]> Mon 23 Sep 2019 11:50:11 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:33230 Mon 23 Sep 2019 10:27:48 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:32466 2+ define loci at which WI papillae form in developing adaxial epidermal transfer cells of Vicia faba cotyledons that are induced to trans-differentiate when the cotyledons are placed on culture medium. We evaluated the hypothesis that vesicle trafficking along a Ca²⁺-regulated remodelled actin network is the mechanism that underpins this outcome. Polarized to the outer periclinal cytoplasm, a Ca²⁺-dependent remodelling of long actin bundles into short, thin bundles was found to be essential for assembling WI papillae but not the underlying uniform wall layer. The remodelled actin network directed polarized vesicle trafficking to sites of WI papillae construction, and a pharmacological study indicated that both exo- and endocytosis contributed to assembly of the papillae. Potential candidates responsible for the Ca²⁺-dependent actin remodelling, along with those underpinning polarized exo- and endocyotosis, were identified in a transcriptome RNAseq database generated from the trans-differentiating epidermal cells. Of most significance, endocytosis was controlled by up-regulated expression of a dynamin-like isoform. How a cycle of localized exo- and endocytosis, regulated by Ca²⁺-dependent actin remodelling, assembles WI papillae is discussed.]]> Mon 23 Sep 2019 10:09:31 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:46193 Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (_apoROS), and cytosolic Ca²⁺. Apoplasmic hydrogen peroxide (_apoH₂O₂) was confirmed as the _apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H₂O₂ biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in _apoH₂O₂ production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of _apoH₂O₂. The findings were consistent with catalase activity constraining the _apoH₂O₂ signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular _apoH₂O₂ signatures that differed during assembly of the UWL and WI papillae.]]> Mon 14 Nov 2022 11:15:50 AEDT ]]>