Wall Ingrowth Development Research Articles

High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

BackgroundTransfer 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.ResultsThe 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.ConclusionsUse 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0483-8) contains supplementary material, which is available to authorized users.

Differential transcriptional networks associated with key phases of ingrowth wall construction in trans-differentiating epidermal transfer cells of Vicia faba cotyledons.

BackgroundTransfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. The ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. However, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood.ResultsAn RNAseq study of an inducible epidermal transfer cell system in cultured 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.ConclusionsPathways 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0486-5) contains supplementary material, which is available to authorized users.

Phloem parenchyma transfer cells in Arabidopsis – an experimental system to identify transcriptional regulators of wall ingrowth formation

In species performing apoplasmic loading, phloem cells adjacent to sieve elements often develop into transfer cells (TCs) with wall ingrowths. The highly invaginated wall ingrowths serve to amplify plasma membrane surface area to achieve increased rates of apoplasmic transport, and may also serve as physical barriers to deter pathogen invasion. Wall ingrowth formation in TCs therefore plays an important role in phloem biology, however, the transcriptional switches regulating the deposition of this unique example of highly localized wall building remain unknown. Phloem parenchyma (PP) TCs in Arabidopsis veins provide an experimental system to identify such switches. The extent of ingrowth deposition responds to various abiotic and applied stresses, enabling bioinformatics to identify candidate regulatory genes. Furthermore, simple fluorescence staining of PP TCs in leaves enables phenotypic analysis of relevant mutants. Combining these approaches resulted in the identification of GIGANTEA as a regulatory component in the pathway controlling wall ingrowth development in PP TCs. Further utilization of this approach has identified two NAC (NAM, ATAF1/2 and CUC2)-domain and two MYB-related genes as putative transcriptional switches regulating wall ingrowth deposition in these cells.

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.

Induction of wall ingrowths of transfer cells occurs rapidly and depends upon gene expression in cotyledons of developing Vicia faba seeds

Abaxial epidermal cells of developing faba bean (Vicia faba) cotyledons are modified to a transfer cell morphology and function. In contrast, the adaxial epidermal cells do not form transfer cells but can be induced to do so when excised cotyledons are cultured on an agar medium. The first fenestrated layer of wall ingrowths is apparent within 24 h of cotyledon exposure to culture medium. The time course of wall ingrowth formation was examined further. By 2 h following cotyledon excision, a 350 nm thick wall was deposited evenly over the outer periclinal walls of adaxial epidermal cells and densities of cytoplasmic vesicles increased. After 3 h in culture, 10% of epidermal cells contained small projections of wall material on their outer periclinal walls. Thereafter, this percentage rose sharply and reached a maximum of 90% by 15 h. Continuous culture of cotyledons on a medium containing 6-methyl purine (an inhibitor of RNA synthesis) completely blocked wall ingrowth formation. In contrast, if exposure to 6-methyl purine was delayed for 1 h at the start of the culture period, the adaxial epidermal cells were found to contain small wall ingrowths. Treating cotyledons for 1 h with 6-methyl purine at 15 h following cotyledon excision halted further wall ingrowth development. We conclude that transfer cell induction is rapid and that signalling and early events leading to wall ingrowth formation depend upon gene expression. In addition, these gene products have a high turnover rate.

Role of light and jasmonic acid signaling in regulating foliar phloem cell wall ingrowth development.

Phloem cells adjacent to sieve elements can possess wall invaginations. The role of light and jasmonic acid signaling in wall ingrowth development was examined in pea companion cells (CCs), Arabidopsis thaliana phloem parenchyma cells (PCs), and in Senecio vulgaris (with ingrowths in both cell types). Features characterized included wall ingrowths (from electron microscopic images), foliar vein density and photosynthetic capacity. In Arabidopsis, wall ingrowths were bulky compared with finger-like invaginations in pea and S. vulgaris. Relative to low light (LL), wall invagination in both CCs and PCs was greater in high light (HL). Treatment with methyl jasmonate in LL had no effect on CCs, but increased PC wall ingrowths. LL-to-HL transfer resulted in significantly less wall ingrowth in the fad7-1 fad8-1 (jasmonate-deficient) Arabidopsis mutant relative to the wild type. These results suggest that chloroplast oxidative status, via chloroplast-derived jasmonates, may modulate phloem structure and function. While CC wall ingrowths facilitate phloem loading by expanding the membrane area available for active uptake, one can speculate that phloem PC ingrowths may have two potential roles: to increase the efflux of sugars and/or protons into the apoplast to augment phloem loading; and/or to protect the phloem against pathogens and/or insects.

Spatial and temporal expression of sucrose transport-related genes in developing cotyledons ofVicia faba L.

Dry-matter accumulation by developing cotyledons of grain legumes includes a mandatory influx of photoassimilates, largely in the form of sucrose, from the seed apoplasm across the plasma membranes of the cotyledon cells. This study examined the temporal and spatial expression of an H+/sucrose symporter, a P-type H+-ATPase, and a sucrose-binding protein (SBP) in cotyledons ofVicia faba L. throughout their development. The flux of dry matter and sucrose symporter activity exhibited identical temporal trends. These were a marked increase during cotyledon expansion to a plateau maintained until cotyledon maturity. Thereafter both parameters declined precipitously. The temporal changes in sucrose symporter activity were accounted for by shifts in its Vmax. Transcript levels of the H+/sucrose symporter followed a similar temporal pattern to the sucrose symporter activity suggesting regulation by gene expression. Equivalent conclusions were drawn for SBP and the H+-ATPase expression during cotyledon expansion. Thereafter, during seed filling, the transcript levels of SBP and H+-ATPase did not closely follow that found for the sucrose symporter. A progressive wave of gene expression in the abaxial epidermal cells spread from the cotyledon region juxtaposed to the non-vascular region of the seed coat at the pole distal from the funicle. The pattern of expression progressed most rapidly along the median longitudinal plane of the cotyledons and more slowly outward to their margins. The densities of SBP and H+-ATPase, inserted into the plasma membranes of the abaxial epidermal cells, increased throughout cotyledon expansion. Gene expression (sucrose symporter) and membrane insertion of the gene products (SBP, H+-ATPase) were closely associated with the initiation and development of wall ingrowths in the abaxial epidermal cells.

Ultrastructure of transfer cells in spontaneous nodules of alfalfa (Medicago sativa)

Spontaneous nodules were formed on the primary roots of alfalfa plants in the absence ofRhizobium. Histologically, these white single-to-multilobed structures showed nodule meristems, cortex, endodermis, central zone, and vascular strands. Nodules were devoid of bacteria and infection threads. Instead, the larger cells were completely filled with many starch grains while smaller cells had very few or none. Xylem parenchyma and phloem companion cells exhibited long, filiform and branched wall ingrowths. The characteristic features of both types of transfer cells were polarity of wall ingrowths, high cytoplasmic density, numerous mitochondria, abundant ribosomes, well-developed nucleus and nucleolus, and vesicles originated from rough endoplasmic reticulum. These results were compared with normal nodules induced byRhizobium. Our results suggest that xylem parenchyma and phloem companion transfer cells are active and probably involved in the short distance transport of solutes in and out of spontaneous nodules. Since younger nodules showed short, papillate, and unbranched wall ingrowths, and older tissue showed elongated, filiform and branched wall ingrowths, the development of wall ingrowths seemed to be gradual rather then abrupt. The occurrence of both type-A and -B wall ingrowths suggests that phloem companion transfer cells may be active in loading and unloading of sieve elements. Since there were no symbiotic bacteria and thus no fixed nitrogen, it is tempting to speculate that xylem parenchyma transfer cells may be re-transporting accumulated carbon from starch grains to the rest of the plant body by loading xylem vessels. Fusion of ER-originated vesicles with wall ingrowth membrane indicated the involvement of ER in the membrane formation for elongating wall ingrowths. Since transfer cells were a characteristic feature of both spontaneous andRhizobium-induced nodules, their occurrence and development is controlled by the genetic make-up of alfalfa plant and not by a physiological source or sink emanating from symbiotic bacteria.

EMBRYO SAC DEVELOPMENT IN SOYBEAN: THE CENTRAL CELL AND ASPECTS OF FERTILIZATION

Development of the soybean central cell and events associated with fertilization are described. Central cell development involves the simultaneous formation of numerous multigrain amyloplasts, development of wall ingrowths, reduction of the cell's major vacuole, and nuclear migration. As these processes reach completion, a phase of lytic activity begins. This activity ultimately results in both the breakdown of most multigrain amyloplasts in the central cell and the reestablishment of the cell's large vacuole. While these processes occur, the egg and synergid cells continue development. Examination of the egg apparatus after plasmogamy but prior to karyogamy reveals several changes. Whereas the synergid containing the pollen tube is degenerate and the distribution of organelles in the persistent synergid has changed, the egg cell with a free sperm nucleus exhibits higher levels of metabolite storage and changes in its wall structure. The walls of the newly formed sporophyte are similar to those of egg apparatus cells (Folsom and Cass, Canadian Journal of Botany 68: 2135–2147 [1990]) in that they seem to lack a middle lamella. This layer becomes apparent only in the two‐celled proembryo. While these changes occur in the egg, zygote, and proembryo, synergids undergo different degradative pathways.

A Light and Electron Microscope Study of the Mature Central Cell and Egg Apparatus of Solanum nigrum L. (Solanaceae)

The central cell and egg apparatus of Solanum nigrum L. were examined to determine the location and extent of wall ingrowth (WI) development as well as the type and distribution of storage products. Wall ingrowths are not found along the embryo sac wall but do project as a single, although interrupted, band of varying thickness from the synergid/central cell and egg/central cell walls. Wall ingrowths projecting from the surface of the synergids extend from just above the hook region to the level of the nucleus. A narrower band projects from the surface of the egg cell and is located opposite the egg vacuole. Mitochondria are closely associated with the wall ingrowths in both the central cell and synergids. The ultrastructural characteristics of the central cell and egg apparatus are similar to those reported for other species. However, the chalazal end of the egg cell is covered only by its plasmalemma: no islands of wall material occur. The plasmalemma of the central cell is consistently withdrawn from the chalazal end of the egg cell, forming one or two small electron-lucent spaces filled with small amounts of fibrillar material. The mature synergids resemble secretory cells and possess abundant dilated cisternae of rough endoplasmic reticulum and numerous mitochondria. No degeneration of the synergid cytoplasm occurred. In longitudinal section the filiform apparatus is anchor shaped and stains intensely for carbohydrates with an outer layer staining for proteins. The major repository for storage products is the central cell where numerous starch-filled amyloplasts accumulate. Small amounts of starch and lipid are present in the egg cell, but the synergids contain only minor amounts of storage lipid in the form of osmiophilic droplets. A hypostase with extensive wall ingrowth development surrounds the antipodal cells.

Zostera capensis setchell: III. Some aspects of wall ingrowth development in leaf blade epidermal cells

The development of wall ingrowths in leaf blade epidermal cells of the marine angiospermZostera capensis was studied by electron microscopy. Prior to the appearance of ingrowths long profiles of endoplasmic reticulum cisternae become arranged peripherally closely following the contours of the walls. The plasmalemma assumes a wavy appearance and in regions where wall ingrowths first start forming (i.e., along the radial, inner tangential and transverse walls) the plasmalemma becomes separated from the walls by an undulating extracytoplasmic space. Small, irregular projections of secondary wall material make their appearance here. Paramural bodies, dictyosomes, endoplasmic reticulum (ER) and possibly also microtubules seem to be closely associated with the initiation and subsequent development of wall projections. As the cells mature, new ingrowths arise in a centrifugal direction along the radial and transverse walls. When wall ingrowths reach a certain stage of their development, mitochondria become strongly polarized towards them and become closely associated with the plasmalemma which ensheaths the ingrowths. There is often also a close association between ER cisternae and the involuted plasmalemma of the wall projections. Initially ingrowths are slender, curved structures, but become more complex as the cells mature. Ingrowths are most extensively developed along the inner tangential and transverse walls. As epidermal cells age there is a loss of wall material from the ingrowths. The probable significance of the formation of wall ingrowths in the epidermal cells is also discussed.

The occurrence and ontogeny of transfer cells associated with lateral roots and root nodules in Leguminosae

Xylem parenchyma transfer cells are present in the stele of the root tissue adjacent to emergent effective root nodules of garden pea (Pisum sativum), red kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), soybean (Glycine max), and mung bean (Vigna radiata), two types of ineffective pea nodules, and emergent lateral roots of pea. The xylem parenchyma transfer cells contain many polyribosomes and mitochondria near the wall ingrowths which are located adjacent to pits in the xylem elements. Pericycle transfer cells also occur in the three types of pea nodules. In effective pea nodules wall ingrowths begin to form in the pericycle cells 5 days after inoculation with Rhizobium leguminosarum; at this stage rhizobia are only present in the root hair but the cortical cells have enlarged and some have undergone mitosis. The wall ingrowths begin to form in the xylem parenchyma cells 7–8 days after inoculation or the approximate time that rhizobia begin to be released from the infection thread. In both instances the wall ingrowths begin to form before the onset of dinitrogen reduction although previous workers have suggested that a flux of nitrogenous compounds (containing fixed N) induces their formation. The development of wall ingrowths in ineffective pea nodules also occurs independently of nitrogen fixation. Similarly, the wall ingrowths located near soybean nodules also begin to develop before the onset of nitrogen fixation.

Structure and Function of Transfer Cells in the Sporophyte Haustorium ofFunaria hygrometricaHedw

Excised sporophytes of the moss Funaria hygrometrica Hedw. absorb externally applied sugar through their basal haustorium. Influx of [3H]sucrose is inhibited by metabolic uncouplers, darkness, and by the photosynthetic inhibitor DCMU. The kinetics of uptake of glucose and sucrose suggest a biphasic mechanism of absorption. Uptake of 3-0-methyl [3H]glucose shows no saturation characteristics and a passive mechanism is indicated. Externally applied glucose is rapidly converted to sucrose. Good retention of products of short-term absorption and metabolism of [3H]glucose was achieved by freeze-substitution. Autoradiography showed dense and uniform labelling of the transfer cells of the haustorium. Kmax values for uptake of sucrose and glucose, expressed in terms of the weight and external surface area of haustorium, are considerably greater than typical values from other plant systems. However, if the surface area amplification that is brought about by the development of wall ingrowths in the transfer cells is taken into account, fluxes per unit area of plasma membrane are reduced into the range of typical values. The hypothesis that the surface area amplification that characterizes transfer cells is related functionally to processes of solute transport is therefore supported by the data.

Aspects of Vascular Anatomy and Differentiation of Vascular Tissues and Transfer Cells in Vegetative Nodes of Wheat

Xylem transfer cells are strongly developed in the departing leaf traces of the mature wheat node. Their differentiation is initiated soon after the appearance of the first tracheary elements in these bundles. and wall ingrowth development reaches its peak just as the leaf to which the bundle belongs becomes fully expanded. It is suggested that the xylem transfer cells play an important role in redirecting solutes travelling in the xylem of the mature leaf to the developing leaves at the shoot apex. It is further suggested that they form an integral part of the normal xylem transpiration pathway, compensating for xylem restrictions and discontinuities in the mature node. Phloem transfer cells also appear very early in the differentiation of the nodal vasculature, although their function remains obscure.

Wall ingrowths in the jacket cells of the antheridia ofAnthoceros laevisL.

As the antheridia of Anthoceros near maturity, wall ingrowths develop along the inner faces of the jacket cells. These cells contain numerous mitochondria and abundant rough endoplasmic reticulum (ER) thus resembling transfer cells described previously from a variety of other anatomical situations. Microbodies, hitherto undescribed in the gametophyte of Anthoceros, also occur in the jacket cells. Since ER and ribosomes are absent from the nearly mature spermatocytes of Anthoceros, production of proteins for the final stages in their development is considered to be a likely function of the jacket cells. The increase in surface area of the cell membrane, brought about by the wall ingrowths, may be an adaptation to facilitate transport of such materials from the jacket cells. Wall ingrowth development may also be related to the retrieval of excess carbohydrate from the maturing spermatocytes. In addition it is suggested that the wall thickenings in the jacket cells of Anthoceros may playa part in the spermatozoid dissemination mechanism.