Glucose and ethylene signalling pathways converge to regulate trans-differentiation of epidermal transfer cells in Vicia narbonensis cotyledons.

Transfer cells are specialized transport cells containing invaginated wall ingrowths that provide an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites where enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, the signal(s) and signalling cascades responsible for initiating their trans-differentiation are poorly understood. In culture, adaxial epidermal cells of Vicia narbonensis cotyledons were induced to trans-differentiate to a transfer cell morphology. Manipulating their intracellular glucose concentrations by transgenic knock-down of ADP-glucose pyrophosphorylase expression and/or culture on a high-glucose medium demonstrated that glucose functioned as a negative regulator of wall ingrowth induction. In contrast, glucose had no detectable effect on wall ingrowth morphology. The effect on wall ingrowth induction of culture on media containing glucose analogues suggested that glucose acts through a hexokinase-dependent signalling pathway. Elevation of an epidermal cell-specific ethylene signal alone, or in combination with glucose analogues, countered the negative effect of glucose on wall ingrowth induction. Glucose modulated the amplitude of ethylene-stimulated wall ingrowth induction by down-regulating the expression of ethylene biosynthetic genes and an ethylene insensitive 3 (EIN3)-like gene (EIL) encoding a key transcription factor in the ethylene signalling cascade. A model is presented describing the interaction between glucose and ethylene signalling pathways regulating the induction of wall ingrowth formation in adaxial epidermal cells.

Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons.

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.

GIGANTEA is a component of a regulatory pathway determining wall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis thaliana.

Transfer cells are specialised transport cells containing invaginated wall ingrowths that generate an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites at which enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, little is known of the molecular mechanisms regulating construction of their intricate wall ingrowths. We investigated the genetic control of wall ingrowth formation in phloem parenchyma transfer cells of leaf minor veins in Arabidopsis thaliana. Wall ingrowth development in these cells is substantially enhanced upon exposing plants to high-light or cold treatments. A hierarchical bioinformatic analysis of public microarray datasets derived from the leaves of plants subjected to these treatments identified GIGANTEA (GI) as one of 46 genes that are commonly up-regulated twofold or more under both high-light and cold conditions. Histological analysis of the GI mutants gi-2 and gi-3 showed that the amount of phloem parenchyma containing wall ingrowths was reduced 15-fold compared with wild-type. Discrete papillate wall ingrowths were formed in gi-2 plants but failed to develop into branched networks. Wall ingrowth development in gi-2 was not rescued by exposing these plants to high-light or cold conditions. In contrast, over-expression of GI in the gi-2 background restored wall ingrowth deposition to wild-type levels. These results indicate that GI regulates the ongoing development of wall ingrowth networks at a point downstream of inputs from environmental signals.

An epidermal-specific ethylene signal cascade regulates trans-differentiation of transfer cells in Vicia faba cotyledons.

*Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and signalling cascades responsible for initiating this trans-differentiation event are poorly understood. We tested the hypothesis that ethylene functions as a key inductive signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons. *Scanning electron microscopy of epidermal cells monitored their propensity for wall ingrowth formation. Spatial and temporal expression profiles of ethylene biosynthetic enzymes and key elements of ethylene signalling cascades (ethylene insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined. *Wall-ingrowth formation responded positively to manipulation of ethylene biosynthesis and perception. It was preceded by a cell-specific burst in ethylene biosynthesis accompanied by a co-localized post-translational up-regulation of VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene production arrested ongoing wall ingrowth development. Wound-induced ethylene in pod walls and seed coats caused an in planta activation of ethylene biosynthetic genes in adaxial epidermal cells that coincidentally formed wall ingrowths. *A cell-specific burst of ethylene biosynthesis functions as an inductive signal initiating and sustaining trans-differentiation to a TC morphology in vitro. These events are reproduced for developing V. faba seeds in planta.

Early gene expression programs accompanying trans-differentiation of epidermal cells of Vicia faba cotyledons into transfer cells.

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Intersection of transfer cells with phloem biology-broad evolutionary trends, function, and induction.

Transfer cells (TCs) are ubiquitous throughout the plant kingdom. Their unique ingrowth wall labyrinths, supporting a plasma membrane enriched in transporter proteins, provides these cells with an enhanced membrane transport capacity for resources. In certain plant species, TCs have been shown to function to facilitate phloem loading and/or unloading at cellular sites of intense resource exchange between symplasmic/apoplasmic compartments. Within the phloem, the key cellular locations of TCs are leaf minor veins of collection phloem and stem nodes of transport phloem. In these locations, companion and phloem parenchyma cells trans-differentiate to a TC morphology consistent with facilitating loading and re-distribution of resources, respectively. At a species level, occurrence of TCs is significantly higher in transport than in collection phloem. TCs are absent from release phloem, but occur within post-sieve element unloading pathways and particularly at interfaces between generations of developing Angiosperm seeds. Experimental accessibility of seed TCs has provided opportunities to investigate their inductive signaling, regulation of ingrowth wall formation and membrane transport function. This review uses this information base to explore current knowledge of phloem transport function and inductive signaling for phloem-associated TCs. The functional role of collection phloem and seed TCs is supported by definitive evidence, but no such information is available for stem node TCs that present an almost intractable experimental challenge. There is an emerging understanding of inductive signals and signaling pathways responsible for initiating trans-differentiation to a TC morphology in developing seeds. However, scant information is available to comment on a potential role for inductive signals (auxin, ethylene and reactive oxygen species) that induce seed TCs, in regulating induction of phloem-associated TCs. Biotic phloem invaders have been used as a model to speculate on involvement of these signals.

Extracellular hydrogen peroxide, produced through a respiratory burst oxidase/superoxide dismutase pathway, directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons.

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing 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.