Associate Professor David McCurdy

© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca 2+ signals generated by spatiotemporal alterations in cytosolic Ca 2+ ([Ca 2+ ] 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 Ca 2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca 2+ signal intensity, by withdrawing extracellular Ca 2+ or blocking Ca 2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca 2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca 2+ by co-operative functioning of plasma membrane Ca 2+ -permeable channels and Ca 2+ -ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca 2+ signal was organized into discrete patches that aligned spatially with clusters of Ca 2+ -permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca 2+ were consistent with inward-directed plumes of elevated [Ca 2+ ] cyt . Plume formation depended upon an alternating distribution of Ca 2+ -permeable channels and Ca 2+ -ATPase clusters. On further inward diffusion, the Ca 2+ plumes coalesced into a uniform Ca 2+ signal. Blocking or dispersing the Ca 2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca 2+ plumes define the loci at which wall ingrowth papillae are deposited.

© 2015 Gao, Jiang, Wing, Jiang and Jackson. All Rights Reserved. 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.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd. Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Ga, one Gß, and one G¿ subunit. However, in addition to the canonical G¿ subunits (class A), plants also possess two unusual, plant-specific classes of G¿ subunits (classes B and C) that have not yet been found in animals. These include G¿ subunits lacking the C-terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub-population of Gß/¿ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C G¿ subunits, which are twice the size of canonical G¿ subunits, with a predicted transmembrane domain and a large cysteine-rich extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C G¿ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular. Significance Statement AGG3 is the prototype of an unusual G¿ subunit of the heterotrimeric G protein complex. It is a type II membrane protein having a cysteine-rich extracellular domain.

© 2015 Nguyen and McCurdy; licensee BioMed Central. Background: Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. Results: The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation. Conclusions: Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.