Hydrophobic protein from soybean (HPS) is present in soybean dust and is an allergen (Gly m 1) that causes asthma in allergic individuals. Past studies have shown that HPS occurs on the seed surface. To determine the microscopic localization of HPS during seed development, monoclonal antibodies to HPS were used to visualize the protein by fluorescence and transmission electron microscopy. Seed coat and endocarp sections were also examined for pectin, cellulose, callose, starch, and protein by histochemical staining. HPS is present in the endocarp epidermal cells at 18 to 28 days post anthesis. At later stages of seed development, HPS occurs in extracellular secretions that accumulate unevenly on the endocarp epidermis and seed surface. HPS is synthesized by the endocarp epidermis and deposited on the seed surface as part of a heterogeneous matrix.

Plants accumulate flavonoids in response to a myriad of environmental challenges, especially when exposed to ultraviolet (UV) radiation or situations causing oxidative stress. However, the origin and nature of the signal triggering their accumulation remain obscure. In this study, a group of flavonoids belonging to the flavone class was identified in Lemna gibba (duckweed). These flavones accumulated upon exposure to UV radiation, low temperature, copper and the photosynthetic electron transport (PET) inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and 1,2-dihydroxyanthraquinone (DHATQ). All of these stressors were also shown to promote PET chain (PETC) reduction; however, in the co-presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or a light regime that oxidized the PETC, flavonoid accumulation ceased. Chloroplast-derived reactive oxygen species (ROS) were not associated with all of the stress conditions that promoted both PETC reduction and flavonoid synthesis, indicating that ROS were not a strict requisite for flavonoid accumulation. Transcripts for the flavonoid biosynthetic genes, chalcone synthase (CHS) and chalcone isomerase, were similarly responsive to the PETC redox state, as were a panel of transcripts revealed by differential display PCR. Collectively, these results provide evidence that PETC redox status is one of the factors affecting flavonoid biosynthesis.

The environmental and seasonal effects on anatomical traits of Pinus taeda L. seedling roots were studied in the laboratory in three contrasting root growth media and also in typical outdoor nursery culture. Growth media with lower water regimen and high penetration resistance caused a reduction in lengths of the white and condensed tannin (CT) zones and acceleration of development of suberin lamellae in the endodermis. As a possible counter to this reduction in zone lengths, second-order laterals were produced closer to the tips of first-order laterals. This suggested there may be an advantage to producing more shorter roots under stressful conditions. Under outdoor nursery conditions (June to mid-December) the white zone was always a rather small part of the root system surface area (4.5% in December), but it dominated as a provider of cortical plasmalemma surface area (CPSA) in contact with modified soil solution (65% in December) because of its live cortex and capacity to increase nearly three fold the amount of CPSA per unit root length. The CT zone always provided most of the total root surface area (80% in December). Although it had no live cortex, a few cells of the CT zone endodermis remained non-suberized passage cells, perhaps giving this major part of the root system some capacity for ion and water absorption. A late summer increase in CPSA was due largely to the rapid production of mycorrhizae. Root systems were capable of very rapid replacement of roots lost due to undercutting and lateral root pruning. The great variation in CPSA per unit root length contained in the white, mycorrhizal and CT zones suggested a capacity to adapt rapidly to changing conditions.

Hypoxia can stimulate the development of a suberized exodermis in aquatic plants; however, its influence on this aspect of terrestrial root development is sparsely documented. To determine the effects of hypoxia on maize (Zea mays cv. Seneca Horizon) roots, seedlings were grown in vermiculite (VERM), aerated hydroponics (AER), stagnant hydroponics with agar (STAG), or aerated hydroponics with agar (AERAG). The endo- and exodermis were examined for wall modifications. Lateral root emergence and aerenchyma formation were documented qualitatively. The endodermal Casparian band formation was unaffected by treatment. Endodermal and exodermal suberin lamella formation was earliest and most extensive in VERM. Suberization, especially in the exodermis of aerated treatments, was depressed in all hydroponic media. In comparison with AER, STAG exodermal lamellae were increased, but endodermal lamellae were decreased. Since the suberized exodermis forms a barrier to radial oxygen loss from roots to the medium, its stimulation in STAG roots (which also developed extensive aerenchyma) would help retain oxygen in the root. The reduction of endodermal lamellae should facilitate oxygen diffusion into the stele. Clearly, the response to environmental conditions is variable within individual cortical cell layers. Additionally, the observed patterns of lamellae, aerenchyma and lateral root development indicate a tight radial co-ordination of root development.

AIMS We review literature and present new observations on the differences among three general patterns of aerenchyma origin and their systematic distributions among the flowering plants, and we clarify terminology on root aerenchyma. SCOPE From our own previous works and some new observations, we have analysed the root cortex in 85 species of 41 families in 21 orders of flowering plants that typically grow in wetlands to determine the characteristic patterns of aerenchyma. FINDINGS A developmental and structural pattern that we term expansigeny, as manifested by honeycomb aerenchyma, is characteristic of all aquatic basal angiosperms (the Nymphaeales) and basal monocots (the Acorales). Expansigenous aerenchyma develops by expansion of intercellular spaces into lacunae by cell division and cell expansion. Schizogeny and lysigeny, so often characterized in recent reviews as the only patterns of root cortex lacunar formation, are present in most wetland plants, but are clearly not present in the most basal flowering plants. CONCLUSION We conclude that expansigeny is the basic type of aerenchyma development in roots of flowering plants and that the presence of expansigenous honeycomb aerenchyma in root cortices was fundamental to the success of the earliest flowering plants found in wetland environments.

Actively growing roots of pouch-grown Pinus banksiana Lamb. are known to have three anatomically distinct zones, i.e., white, condensed tannin, and cork (in order of increasing distance from the root tip). Roots of pouch and pot-grown Pinus taeda L., and field-grown P. banksiana also develop these three zones. The terminal region of a dormant root resembles the condensed tannin zone, with the addition of a suberized metacutis partially surrounding the apical meristem. White roots are anatomically suited for efficient ion uptake due to the presence of a living cortex. The condensed tannin zones of both growing and dormant roots have a dead cortex but retain passage cells in their endodermal layers, through which some ion uptake could occur. The effect of the maturation from white to condensed tannin zone on water uptake is difficult to predict, but some uptake would occur through the endodermal passage cells. In the young cork zone, no ion and little water absorption should occur. The discrepancies between results of separate anatomical and physiological investigations of tree roots need to be resolved by correlative studies incorporating both approaches in individual experiments.

Roots of virtually all vascular plants have an endodermis with a Casparian band, and the majority of angiosperm roots tested also have an exodermis with a Casparian band. Both the endodermis and exodermis may develop suberin lamellae and thick, tertiary walls. Each of these wall modifications has its own function(s). The endodermal Casparian band prevents the unimpeded movement of apoplastic substances into the stele and also prevents the backflow of ions that have moved into the stele symplastically and then were released into its apoplast. In roots with a mature exodermis, the barrier to apoplastic inflow of ions occurs near the root surface, but prevention of backflow of ions from the stele remains a function of the endodermis. The suberin lamellae protect against pathogen invasion and possibly root drying during times of stress. Tertiary walls of the endodermis and exodermis are believed to function in mechanical support of the root, but this idea remains to be tested. During stress, root growth rates decline, and the endodermis and exodermis develop closer to the root tip. In two cases, stress is known to induce the formation of an exodermis, and in several other cases to accelerate the development of both the exodermis and endodermis. The responses of the endodermis and exodermis to drought, exposure to moist air, flooding, salinity, ion deficiency, acidity, and mechanical impedance are discussed.

Adventitious roots of marsh-grown Pontederia cordata were examined to determine cortical development and structure. The innermost layer of the ground meristem forms the endodermis and aerenchymatous cortex. The outermost layer of the early ground meristem undergoes a precise pattern of oblique and periclinal cell divisions to produce a single or double layer of prohypodermis with an anchor cell for each radial file of aerenchyma cells. At maturity, endodermal cell walls are modified only by narrow Casparian bands. The central regions of the ground meristem become proaerenchyma and exhibit asymmetric cell division and expansion. They produce an aerenchymatous zone with barrel-shaped large cells and irregularly shaped small cells traversing the aerenchyma horizontally along radii; some crystalliferous cells with raphides are present in the aerenchyma. The walls of the hypodermis are modified early by polyphenols. The outermost layer of the hypodermis later matures into an exodermis with Casparian bands that are impermeable to berberine, an apoplastic tracer dye. The nonexodermal layer(s) of the hypodermis has suberin-modified walls. Radial files of aerenchyma are usually connected by narrow protuberances near their midpoints, the aerenchyma lacunae having been produced by expansion of cells along walls lining intercellular spaces. We are terming this type of aerenchyma development, which is neither schizogenous nor lysigenous, "differential expansion."

. The development of tap root anatomical features was investigated in seedlings of loblolly pine ( Pinus taeda L.) under both pot and pouch growth regimes. The roots possessed the three anatomical zones previously observed in jack pine ( Pinus banksiana Lamb) and Eucalyptus pilularis Sm. – white, condensed tannin (CT), and cork – suggesting that this developmental sequence is preserved over species and growth conditions. Xylem development was centripetal and similar to that found earlier in P. sylvestris . Tracheids with lignified, secondary walls were detected distal to the point of endodermal Casparian band deposition. However, tests for ability to conduct fluid indicated that the protoxylem was capable of transport only proximal to the Casparian bands. Detailed examination of suberin lamella deposition in the endodermis demonstrated that passage cells were present through the white and CT zones. Progressive, centripetal cortical death in the CT zone did not include the endodermis, which remained alive until the cork layer formed, at which point the endodermis was crushed. Therefore, passage cells remain as functional portals for nutrient and water uptake in the CT zone even though the central cortex is dead. Tracer tests indicated that the endodermis provides an apoplastic barrier to tracer diffusion into the stele and that this function was taken over by the young cork layers. Results of this study point to a strong role for the endodermis in the regulation of nutrient and water uptake until the maturation of the first cork layer.

Tissues in adventitious roots of Hydrocharis morsus-ranae L. developed from a four-tiered apical meristem. A set of periclinal divisions in the outermost layer of the ground meristem produced a hypodermis, which was normally uniformly biseriate. Aerenchyma formed from the adjacent inner layer of the cortex by a series of cell divisions and cell lyses; three- to five-celled, radial aerenchyma strands formed by periclinal divisions in radial cell files 0.3-5 mm behind the apex. Intervening cells underwent anticlinal and periclinal divisions followed by cell lyses within 1 mm of the apex to produce air spaces. Aerenchyma formation in this species is unusual and presents a unique system suitable for a study of developmentally programmed cell death in parenchyma cells. The endodermis formed a complete Casparian band about 10 mm behind the root apex and did not develop further; it had neither suberin lamellae nor secondary walls. The hypodermis was parenchymatous and was without Casparian bands, suberin lamellae, and secondary walls. Following acid digestion, the wavy walls of the endodermis and the walls of the epidermis remained.Key words: aerenchyma, cell death, endodermis, Hydrocharis, hypodermis, root development.

The development of the endodermis and hypodermis in adventitious roots of Typha angustifolia L. and Typha glauca Godr. was followed from the apical meristem to full maturity. The endodermis was typical, developing a thin Casparian band near the root tip, followed by suberin lamellae and asymmetric, secondary, lignified walls (C-type at maturity). Passage cells were present at an intermediate stage but eventually disappeared when all cells developed lamellae and secondary walls. The hypodermis was multiple (four to six layers at maturity) and began differentiating near the root tip. Here, the radial and transverse walls of the outermost layer did not dissolve in strong acid and the former were wavy in the longitudinal direction, both features characteristic of a Casparian band, but these walls were permeable to berberine. No other indication of a wall modification was seen for 3 weeks, at which time the root had become determinate and aerenchyma was beginning to form in the midcortex. Casparian bands, which were impermeable to berberine, matured in the hypodermis; thus, it proved to be an exodermis. Different forms of Casparian band were detected: one was typical and occupied the radial and transverse walls of the outermost layer, but others were novel and included tangential walls, often forming an H-shaped structure (as seen in cross section of the root). We propose calling the latter type an H-type Casparian band. It functioned as an apoplastic barrier to berberine applied either externally or internally by injection into the cortical aerenchyma. Following maturation of the Casparian band, the outer two layers of the exodermis soon produced suberin lamellae. These continued to be deposited in a centripetal pattern until they were found in all layers of the multiple exodermis. Development of the suberin wall modifications correlates with the development of the aerenchyma and may play a role in preventing gas exchange between the root and the rhizosphere. Later, all exodermal cells produced lignified, secondary walls. These were asymmetric in the outermost and innermost layers of the hypodermis (like the C-type endodermis); eventually, all layers had Casparian wall materials. Thus, the mature hypodermis consisted of two to six layers of exodermis, except at the tips of determinate roots where the exodermis was uniseriate with typical Casparian bands.Key words: cattail, endodermis, exodermis, hypodermis, roots, Typha.

When the basal zones of 4-d-old hydroponically grown maize (Zea mays L. cv. Seneca Horizon) roots were exposed to moist air for 2 d, the development of both endodermis and exodermis was affected. In the endodermis, Casparian bands enlarged and more cells developed suberin lamellae. The most striking effect was seen in the exodermis, In submerged controls, only 4% of the cells had Casparian bands, whereas in root regions exposed to air, 93% developed these structures. Similarly, in submerged roots 11% of the exodermal cells had either developing or mature suberin lamellae compared with 92% in the air-treated region. The majority of epidermal cells remained alive in the zone exposed to air. Some cell death had occurred earlier in the experiment when the seedlings were transferred from vermiculite to hydroponic culture. The precise stimulus(i) associated with the air treatment which led to accelerated development in both endodermis and exodermis is as yet unknown.

Water moves radially through the root in response to the tension generated by the transpiration stream. This movement occurs through both the cell walls and the protoplasts of the cells intervening between the soil solution and the lumena of the tracheary elements. The mechanism of movement is commonly believed to be diffusion in both these compartments. In the present study, we applied the apoplastic, fluorescent tracer, berberine, to roots of three dicotyledonous (Helianthus annuus L. cv. Mammoth Russian, Phaseolus vulgaris L. cv. Kinghorn wax, and Phaseolus aureus Roxb.) and four monocotyledonous species (Triticum aestivum L., Hordeum vulgare L., Zea mays L. cv. Seneca Chief, and Allium cepa L. cv. Ebeneezer). The tracer was precipitated in place by potassium thiocyanate. The entry of berberine into the main roots of the monocotyledonous species was limited, and no conclusions could be drawn about its movement. Tracer entered more readily into the main roots of dicotyledonous species and its movement by diffusion (in excised roots) was characterized by an evenly advancing diffusion ring in the cortex. However, when short treatment times were used for transpiring plants, some berberine was moved across the cortex by solvent drag, resulting in the formation of isolated crystals near the endodermis in advance of the diffusion ring. The phenomenon of solvent drag, in turn, is indirect evidence for movement of water by bulk flow in the cortical cell walls. Whether or not bulk flow also occurred in lateral roots could not be determined since the narrow width of the cortex and the high permeability of the walls to berberine resulted in very fast progression of the diffusion ring.

The exodermal Casparian band in corn (Zea mays L.) was first seen 10 mm distal to the kernel 4 days after planting. From its inception, the band usually occupied most of the radial wall (as seen in a cross section of the root). Subsequent maturation of the band around the root was asynchronous into the region of emerging lateral roots. Thus, a continuous apoplastic barrier would have been absent over much of the young root surface. Suberin lamellae development was also asynchronous, as these structures formed in those cells which had Casparian bands. Frequently, a lamella was initially deposited in patches, progressing centripetally until a continuous lipid layer was formed around the cell protoplast. Many instances of band plasmolysis (typical of the endodermis) were observed in the developing uniform exodermis. It could occur in cells with no detectable Casparian bands, suggesting that the tight connection between the plasmalemma and the wall that causes this phenomenon is not due to hydrophobic attractions. The results are consistent with the idea that there are strong attractions between proteins of the membrane and wall in the region of the Casparian band. The tight connection between the plasmalemma and the wall was broken during the later stages of suberin lamella development. Key words: Zea mays L., Poaceae, band plasmolysis, exodermis, Casparian band, suberin lamella.

Forty-three species of seedless vascular plants were assessed for modifications to root cortical cell walls. All species except Lycopodium had an endodermis with distinct Casparian bands. Experiments with the apoplastic tracer berberine hemisulfate showed that walls of all root cortical cells in the two Lycopodium species tested were permeable to this tracer. Although most species examined lacked a hypodermis several Equisetum species had a hypodermis with modified walls. Three Selaginella species had distinct Casparian bands in this cortical cell layer. This layer, therefore, is an exodermis in Selaginella and its presence limited the inward diffusion of the apoplastic tracer berberine hemisulfate.

Passage cells frequently occur in the endodermis and exodermis but are not ubiquitous in either layer. Passage cells occur in the form of short cells in the dimorphic type of exodermis. In both layers, Casparian bands are formed in all cells, but the subsequent development of suberin lamellae and thick, cellulosic walls are delayed or absent in the passage cells. Available evidence suggests that passage cells of the endodermis are important for the transfer of calcium and magnesium into the stele and thus into the transpiration stream. They become the only cells which present a plasmalemma surface to the soil solution (and are thus capable of ion uptake) when the epidermis and central cortex die. This occurs naturally in some herbaceous and woody species and is known to be promoted by drought. Most evidence indicates that the development of suberin lamellae in both the endodermis and exodermis increases the resistance of the root to the radial flow of water. Passage cells thus provide areas of low resistance for the movement of water, and the position of these cells in the endodermis (i.e., in close proximity to the xylem) is explained in terms of function. Exodermal passage cells have a cytoplasmic structure suggesting an active role in ion uptake. This may be related to the tendency of the epidermis to die, leaving the passage cells as the only ones with their membranes exposed to the soil solution. Passage cells in the exodermis attract endomycorrhizal fungi while those in the endodermis do not. It is clear that passage cells of the endodermis and exodermis play a variety of roles in the plant root system.

The alkaloid berberine is useful as a mobile apoplastic tracer. It is readily precipitated by thiocyanate, forming bright yellow, needle-like, fluorescent crystals. When berberine hemisulphate and potassium thiocyanate are applied sequentially to plant tissue, the crystals form in unmodified walls and in the lumina of dead cells such as tracheary elements. Lignified and suberized walls stain with berberine but do not develop crystals. Regions of the plant that have been penetrated by the chemicals can be located by preparing freehand sections, mounting them in potassium thiocyanate to inhibit dissolution of the crystals, and examining them with a fluorescence microscope. When the two chemicals are sequentially introduced into the xylem of onion, corn, and broad bean roots, the tracer does not pass the endodermal Casparian band, indicating that the system traces apoplastic pathways. The chemical concentrations that produce sufficient crystals so that berberine can be used as an apoplastic tracer in primary roots are 0.05% berberine hemisulphate and 0.09 M potasium thiocyanate. These concentrations were not toxic to cells of onion bulb epidermis. They reduced the growth rates of corn and broad bean roots but did not kill them. Berberine–thiocyanate is a useful apoplastic tracer provided care is taken not to exceed the limits of berberine toxicity to the tissue. Key words: apoplastic tracer, berberine hemisulphate, potassium thiocyanate, roots, toxicity tests, corn, onion, broad bean.

The apoplastic permeability of regions near the root tips of two nonexodermal species (Vicia faba L. and Pisum sativum L.) and three exodermal species (Zea mays L., Allium cepa L., and Helianthus annuus L.) was investigated with a berberine – thiocyanate tracer procedure. In regions of nonexodermal roots where the endodermis was mature, the walls of the epidermis and cortex were permeable; inward diffusion of the tracers was arrested at the endodermis. In regions of exodermal roots where the exodermis was mature, the apoplast was permeable only up to the anticlinal walls of the exodermis; in regions where the exodermis was immature but the endodermis was mature, the cortex was permeable up to the Casparian band of the endodermis. In root tips of both exodermal and nonexodermal species, berberine penetration into the meristem was greatly restricted. In some species there were additional areas adjacent to the meristem into which berberine movement was similarly restricted. It is concluded that even though root apices lack suberized structures, these zones are nevertheless partially apoplastically isolated from the external environment. Key words: apoplastic transport, berberine, fluorescent tracer, root tips.