Discovering Roles of Calcium and Cortical Microtubules in Directing Ingrowth Wall Formation During Trans-differentiation of Epidermal Cells to a Transfer Cell Morphology

Research Doctorate - Doctor of Philosophy in Biological ScienceTransfer cells (TCs) are plant cells specialized for membrane transport of nutrients. Characterized by an ingrowth wall, ensheathed by an amplified plasma membrane enriched with nutrient transporters, TCs are located in nutrient transport ‘bottlenecks’ to facilitate apoplasmic/symplasmic nutrient exchange and hence play an important role in plant growth. Observed in all plant taxonomic groups, TCs trans-differentiate in response to various developmental and environmental cues. Despite their biological significance, underlying mechanisms inducing TC trans-differentiation are poorly understood. Investigation of inductive signalling cascades leading to TC trans-differentiation requires experimental access to a manipulable system with a large population of uncommitted cells that can be induced to undergo TC trans-differentiation. An excellent experimental platform meeting these criteria is provided by Vicia faba cotyledons. In planta, abaxial epidermal cells of V. faba cotyledons trans-differentiate into TCs during seed development. Their adaxial epidermal cells can be induced to trans-differentiate into functional TCs when cotyledons are cultured. Based on these facts, we employed cultured V. faba cotyledons as our experimental system to investigate signalling cascades that lead to TC trans-differentiation. Ingrowth wall development in adaxial epidermal cells of cultured V. faba cotyledons involves deposition of a uniform wall layer on their original outer periclinal walls upon which localized papillate wall ingrowths (WIs) are formed. Ethylene and reactive oxygen species (ROS) have been identified as components of a signalling cascade leading to ingrowth wall formation. However, signals downstream of ethylene and ROS contributing to WI deposition remain unknown. Based on understanding of signals regulating polarized tip growth, Ca2+ was hypothesized to be an important component in signalling cascades leading to TC trans-differentiation. In addition, we hypothesized that cortical microtubules (CMTs) function to organize a localized cellulose microfibril scaffold to construct WIs. Thus, in this thesis, studies are described that test the potential roles of Ca2+ signalling and CMTs, as well as their potential interactions with ethylene and ROS, in regulating ingrowth wall formation during TC trans-differentiation. Visualizing and quantifying WI formation in adaxial epidermal cells with scanning electron microscopy (SEM), we found that extracellular Ca2+ is needed for WI development. The Ca2+ taken into the cell generates a cytosolic Ca2+ signal, polarized to the outer periclinal cytosol of epidermal cells. The signal was detected by a pre-loaded Ca2+ sensitive probe, Oregon Green 488 BAPTA-1 AM ester and confocal laser scanning microscopy. Responses to Ca2+ channel blockers demonstrated that generation and maintenance of the polarized Ca2+ signal depended upon co-operative interaction between Ca2+ influx across the plasma membrane through L-type Two Pore Ca2+ channels matched by an equally rapid Ca2+ efflux from the epidermal cell cytosol. Ca2+ efflux is presumably mediated by Ca2+-ATPase and Ca2+/H+ antiporters. However, neither estimates of cytosolic free Ca2+ concentration nor polarity was altered by treating cotyledons with inhibitors of these Ca2+ effluxers. Clusters of Ca2+ channels were localized to the outer lateral polar domains of epidermal cell plasma membranes and were shown to be responsible for shaping the Ca2+ signal into plumes when entering epidermal cell cytosol. Significantly, the densities and diameters of both the Ca2+ channel clusters and Ca2+ plumes were found to approximate those of papillate WIs. Dissipating the Ca2+ plumes with a Ca2+ ionophore, A23187, inhibited WI deposition. This finding demonstrates that the Ca2+ plumes determine loci for WI formation. In contrast, ROS regulated deposition of the ingrowth wall by acting directly on uniform wall formation and indirectly through Ca2+ channel activity to generate Ca2+ plumes that determine sites of WI deposition. Generation of the Ca2+ signal was shown to be under a dual regulation by ethylene and ROS. To investigate this regulation further, we cloned a Vicia faba two pore Ca2+ channel gene (VfTPC1), the spatio-temporal expression pattern of which was TC-specific. This renders VfTPC1 as a strong candidate for generating the Ca2+ plumes. Attempts were made to characterize intracellular localization of VfTPC1 by immunocytochemistry. However an epitope-specific signal above background could not be detected using an antibody raised against an unique peptide sequence of VfTPC1. Using VfTPC1 as a surrogate for Ca2+ channels supporting the Ca2+ influx generating the Ca2+ signal, intracellular ROS was found to upregulate Ca2+ channel activity at a transcriptional level. In addition, ethylene and ROS upregulated Ca2+ channel activity post-translationally. Combined with previous studies showing that Ca2+ affects ROS production that in turn feeds back on ethylene biosynthesis, an ethylene-ROS-Ca2+ signal hub was identified as the primary cohort of signals regulating induction of trans-differentiation leading to a TC morphology. The Ca2+ signal was shown to be transduced though a Ca2+/Calmodulin (Ca2+/CaM) pathway. On TC induction, parallel-aligned CMT arrays initially became randomized to form a randomized matrix. Concurrent with WI deposition, circular non-fluorescent areas arise within the tubulin network surrounded by CMT bundles forming ‘collars’. The fluorescence intensity of this latter matrix was diminished indicating depletion of CMTs. The non-fluorescent areas were of a diameter that approximated those of WIs and their occurrence followed the same temporal and spatial patterns as WI formation. We tested the causality of the observed interrelationship between CMT organization and WI deposition by stabilizing or depolymerizing CMTs and by blocking WI formation using inhibitors of inductive signals that initiate TC trans-differentiation. WI deposition was not affected by manipulating CMTs. Similarly, blocking WI formation depressed the appearance of ‘collars’ but had no obvious affect on randomization of CMT arrays. We concluded from these results that CMTs have no role to play in WI formation. We predict that appearance of ‘collars’ in the randomized CMT arrays result from papillate WI intrusion into the epidermal cell cytoplasm forcing the CMT bundles to bend around the developing WIs. Interestingly, when CMTs are depolymerized by oryzalin, aberrant clumps of coalescing WI papillae were observed in a small proportion of adaxial epidermal cells. Altering the timing of cotyledon exposure to oryzalin demonstrated that the conditions leading to aberrant WI formation were established within 3 h of cotyledon culture and hence precedes appearance of WIs. This finding indicated that CMTs might have some regulatory role during the early phase of ingrowth wall formation. Overall the body of work reported in this thesis has increased our understanding of TC trans-differentiation by identifying Ca2+ as a key signal, which interacts with ethylene and ROS, to determine loci for WI formation. In conjunction with previous research findings, we develop a model describing signalling pathways and their interactions leading to induction of TC trans-differentiation in adaxial epidermal cells of V. faba cotyledons. We evaluate whether such a signalling pathway is consistent with what is known of in planta TC trans-differentiation across a number of morphological and physiological contexts. The dissertation concludes by identifying several lines of profitable enquiry for future investigation to further unravel mechanisms regulating trans-differentiation to a TC morphology