Carol A. Peterson

Efficient lipid staining in plant material with Sudan red 7B or Fluorol yellow 088 in polyethylene glycol-glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.

A berberine-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue

SummaryA fluorescent staining procedure to detect suberin, lignin and callose in plants has been developed. This procedure greatly improves on previous methods for visualizing Casparian bands in root exodermal and endodermal cells, and performs equally well on a variety of other plant tissues. Berberine was selected as the most suitable replacement forChelidonium majus root extract after comparing the staining properties of the extract with those of four of its constituent alkaloids. Aniline blue counterstaining efficiently quenched unwanted background fluorescence and nonspecific berberine staining, while providing a fluorochrome for callose. When used with multichambered holders which allow simultaneous processing of freehand sections, this efficient staining procedure facilitates morphological studies involving large numbers of samples.

Root Endodermis and Exodermis: Structure, Function, and Responses to the Environment

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.

Transport of Water and Solutes across Maize Roots Modified by Puncturing the Endodermis (Further Evidence for the Composite Transport Model of the Root)

The effects of puncturing the endodermis of young maize roots (Zea mays L.) on their transport properties were measured using the root pressure probe. Small holes with a diameter of 18 to 60 [mu]m were created 70 to 90 mm from the tips of the roots by pushing fine glass tubes radially into them. Such wounds injured about 10–2 to 10–3% of the total surface area of the endodermis, which, in these hydroponically grown roots, had developed a Casparian band but no suberin lamellae. The small injury to the endodermis caused the original root pressure, which varied from 0.08 to 0.19 MPa, to decrease rapidly (half-time = 10–100 s) and substantially to a new steady-state value between 0.02 and 0.07 MPa. The radial hydraulic conductivity (Lpr) of control (uninjured) roots determined using hydrostatic pressure gradients as driving forces was larger by a factor of 10 than that determined using osmotic gradients (averages: Lpr [hydrostatic] = 2.7 x 10–7 m s-1 MPa-1; Lpr [osmotic] = 2.2 x 10–8 m s-1 MPa-1; osmotic solute: NaCl). Puncturing the endodermis did not result in measurable increases in hydraulic conductivities measured by either method. Thus, the endodermis was not rate-limiting root Lpr: apparently the hydraulic resistance of roots was more evenly distributed over the entire root tissue. However, puncturing the endodermis did substantially change the reflection ([sigma]sr) and permeability (Psr) coefficients of roots for NaCl, indicating that the endodermis represented a considerable barrier to the flow of nutrient ions. Values of [sigma]sr decreased from 0.64 to 0.41 (average) and Psr increased by a factor of 2.6, i.e. from 3.8 x 10–9 to 10.1 x 10.-9 m s-1(average). The roots recovered from puncturing after a time and regained root pressure. Measurable increases in root pressure became apparent as soon as 0.5 to 1 h after puncturing, and original or higher root pressures were attained 1.5 to 20 h after injury. However, after recovery roots often did not maintain a stable root pressure, and no further osmotic experiments could be performed with them. The Casparian band of the endodermis is discontinuous at the root tip, where the endodermis has not yet matured, and at sites of developing lateral roots. Measurements of the cross-sectional area of the apoplasmic bypass at the root tip yielded an area of 0.031% of the total surface area of the endodermis. An additional 0.049% was associated with lateral root primordia. These areas are larger than the artificial bypasses created by wounding in this study and may provide pathways for a “natural bypass flow” of water and solutes across the intact root. If there were such a pathway, either in these areas or across the Casparian band itself, roots would have to be treated as a system composed of two parallel pathways (a cell-to-cell and an apoplasmic path). It is demonstrated that this “composite transport model of the root” allows integration of several transport properties of roots that are otherwise difficult to understand, namely (a) the differences between osmotic and hydrostatic water flow, (b) the dependence of root hydraulic resistance on the driving force or water flow across the root, and (c) low reflection coefficients of roots.

Soybean Root Suberin: Anatomical Distribution, Chemical Composition, and Relationship to Partial Resistance to Phytophthora sojae

Soybean (Glycine max L. Merr.) is a versatile and important agronomic crop grown worldwide. Each year millions of dollars of potential yield revenues are lost due to a root rot disease caused by the oomycete Phytophthora sojae (Kaufmann & Gerdemann). Since the root is the primary site of infection by this organism, we undertook an examination of the physicochemical barriers in soybean root, namely, the suberized walls of the epidermis and endodermis, to establish whether or not preformed suberin (i.e. naturally present in noninfected plants) could have a role in partial resistance to P. sojae. Herein we describe the anatomical distribution and chemical composition of soybean root suberin as well as its relationship to partial resistance to P. sojae. Soybean roots contain a state I endodermis (Casparian bands only) within the first 80 mm of the root tip, and a state II endodermis (Casparian bands and some cells with suberin lamellae) in more proximal regions. A state III endodermis (with thick, cellulosic, tertiary walls) was not present within the 200-mm-long roots examined. An exodermis was also absent, but some walls of the epidermal and neighboring cortical cells were suberized. Chemically, soybean root suberin resembles a typical suberin, and consists of waxes, fatty acids, ω-hydroxy acids, α,ω-diacids, primary alcohols, and guaiacyl- and syringyl-substituted phenolics. Total suberin analysis of isolated soybean epidermis/outer cortex and endodermis tissues demonstrated (1) significantly higher amounts in the endodermis compared to the epidermis/outer cortex, (2) increased amounts in the endodermis as the root matured from state I to state II, (3) increased amounts in the epidermis/outer cortex along the axis of the root, and (4) significantly higher amounts in tissues isolated from a cultivar (‘Conrad’) with a high degree of partial resistance to P. sojae compared with a susceptible line (OX760-6). This latter correlation was extended by an analysis of nine independent and 32 recombinant inbred lines (derived from a ‘Conrad’ × OX760-6 cross) ranging in partial resistance to P. sojae: Strong negative correlations (−0.89 and −0.72, respectively) were observed between the amount of the aliphatic component of root suberin and plant mortality in P. sojae-infested fields.

Lateral hydraulic conductivity of early metaxylem vessels in Zea mays L. roots

The hydraulic conductivity of the lateral walls of early metaxylem vessels (Lpx in m · s−1 · MPa−1) was measured in young, excised roots of maize using a root pressure probe. Values for this parameter were determined by comparing the root hydraulic conductivities before and after steam-ringing a short zone on each root. Killing of living tissue virtually canceled its hydraulic resistance. There were no suberin lamellae present in the endodermis of the roots used. The value of Lpx ranged between 3 · 10−7 and 35 · 10−7 m · s−1 · MPa−1 and was larger than the hydraulic conductivity of the untreated root (Lpr = 0.7 · 10−7 to 4.0 · 10−7 m · s−1 · MPa−1) by factor of 3 to 13. Assuming that all flow through the vessel walls was through the pit membranes, which occupied 14% of the total wall area, an upper limit of the hydraulic conductivity of this structure could be given(Lppm=21 · 10−7 to 250 · 10−7 m · s−1 · MPa−1). The specific hydraulic conductivity (Lpcw) of the wall material of the pit membranes (again an upper limit) ranged from 0.3 · 10−12 to 3.8 · 10−12 m2 · s−1 · MPa−1 and was lower than estimates given in the literature for plant cell walls. From the data, we conclude that the majority of the radial resistance to water movement in the root is contributed by living tissue. However, although the lateral walls of the vessels do not limit the rate of water flow in the intact system, they constitute 8–31% of the total resistance, a value which should not be ignored in a detailed analysis of water flow through roots.

Structure and permeability of the fungal sheath in thePisonia mycorrhiza

SummaryThe tracer Cellufluor has been used to test the apoplastic permeability of the fungal sheath inPisonia grandis R. Br. mycorrhizas. In the tip region in the immediate vicinity of the root cap, where the sheath is not yet fully differentiated, Celluflor penetrates as far as the root epidermal cells. Behind this (i.e. just proximal to it) in differentiated regions, where the ultrastructure of both the root and fungal cells indicates that the mycorrhiza is likely to be functionally active, the sheath is impermeable to Cellufluor. During the development and differentiation of the sheath, the interhyphal spaces become filled with extracellular material. In the outer and middle regions this becomes electron opaque after fixation and staining. It is proposed that the dramatic decrease in apoplastic permeability over a short distance back from the root apex as the fungal sheath differentiates results from secretion of extracellular material by the fungus and its modification by deposition of phenolic substances. The symplastic pathway within the fungus may be very important for radial transfer of materials across the sheath. Blockage of the sheath apoplast could provide a sealed apoplastic compartment at the fungus-root interface, with resulting increase in efficiency of transfer between partners. The implications of these observations are discussed in relation to radial transfer across the sheath and transfer between partners in sheathing mycorrhizas in general.

Pine root structure and its potential significance for root function

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.

A New Fluorescent Test for Cell Vitality Using Calcofluor White M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.

Ontogeny and anatomy of lateral roots

Lateral roots arise endogenously from other roots and increase the absorptive surface of the plant. Sometimes referred to as secondary roots, lateral roots ultimately mimic to a large extent, the structure of the root from which they originate. Since most lateral roots are initiated some distance basipetal to the apical meristem, differentiated cells must become reprogrammed to give rise to the initials of lateral root primordia. Subsequently, these initials divide and enlarge in very precise patterns to organize a new organ recognizable as a root. This chapter will consider the structural aspects of lateral root initiation as well as tissue involvement and reorganization in the parent root associated with this process. In addition, the development of some specialized lateral roots will be considered. The most thorough previous treatments of the structural aspects of lateral root formation can be found in Van Tieghem and Douliot [1], Von Guttenberg [2] and McCully [3].