Suberin lamellae in the hypodermis of maize (Zea mays) roots; development and factors affecting the permeability of hypodermal layers

Abstract The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30–50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 μn^?2) as in the junction between the epidermis and hypodermis (0.54 μm^?2). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and ₈₆Rb were low (circa 10^?5mms^?1) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O₂) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.     

Sucrose efflux and export from the maize scutellum*

Abstract During incubation of maize scutellum slices in fructose, there was an efflux of sucrose. Efflux was constant for at least 4 h at fructose concentrations of 70 or 100 mol m^?3. Efflux was increased by EDTA, and decreased by Ca²⁺. Efflux was independent of pH after EDTA treatment, but increased from untreated slices when the pH was lowered from 7 to 4. Uranyl ion and PCMBS (p-chloro-mercuribenzenesulfonic acid) abolished sucrose uptake, but were only weak inhibitors of sucrose efflux. These results are consistent with efflux occurring by simple diffusion through aqueous pores, but they do not rule out facilitated diffusion. Rates of sucrose export from the scutellum to the root shoot axis were estimated from measurements of axis respiration and dry weight gain. Sucrose efflux from scutellum slices was only 14-22% of the export rate. Sucrose efflux from the whole scutellum was only 3-4% of the export rate. It is concluded that the observed efflux is from leaky cells and does not represent sucrose on the way to the phloem along a path that includes the apoplast. These results support the idea that the path for sucrose from parenchyma cell to sieve tube in the maize scutellum is entirely symplastic.     

Sodium-induced calcium deficiency in salt-stressed corn

Abstract The effect of the Na⁺/Ca²⁺ ratio in the root media on salt-stressed corn (Zea mays L. cvs DeKalb XL-75 and Pioneer 3906) was determined in greenhouse experiments. Plants grown in a complete nutrient solution salinized with 86.5 mol m^?3 NaCl exhibited severe Ca²⁺ deficiency symptoms at the four-leaf stage. The symptoms disappeared when part of the NaCl was replaced with 10 mol m^?3 CaCl₂ (Na⁺/Ca²⁺ molar ratio = 5.7). Salt stress at an iso-osmotic potential of ?0.4 MPa substantially decreased shoot growth at all solution Na⁺/Ca²⁺ ratios from 34.6 to 0.26. However, the dry weights of blades at 26 d of age were much less when plants were salinized with NaCl alone, particularly that of DeKalb XL-75 which was more susceptible to Na-induced Ca²⁺ deficiency than was Pioneer 3906. The growth of sheaths was similarity reduced by sail stress at all Na⁺/Ca²⁺ ratios. The symptoms of Ca²⁺ deficiency were correlated with low Ca²⁺ concentrations in the leaf tissue. Ca²⁺ concentrations in the developing blades of NaCl-stressed plants were much lower than in control plants. As the Na⁺/Ca²⁺ ratio in the solution was decreased, Ca²⁺ levels increased in both the blades and sheaths while Na⁺ concentrations greatly decreased. DeKalb XL-75 was much less effective than Pioneer 3906 in restricting the uptake of Na⁺. The results clearly indicate that NaCl stress may cause lesions and unique plant responses that are not manifested on agronomic plants grown on saline soils.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

The influence of calcium and pH on growth in primary roots of Zea mays

Hasenstein, K. H. and Evans, M. L. 1988. The influence of calcium and pH on growth in primary roots of Zea mays. - Physiol. Plant. 72: 466–470.
We investigated the interaction of Ca²⁺ and pH on root elongation in Zea mays L. cv. B73 × Missouri 17 and cv. Merit. Seedlings were raised to contain high levels of Ca²⁺ (HC, imbibed and raised in 10 m M CaCl₂) or low levels of Ca²⁺ (LC, imbibed and raised in distilled water). In HC roots, lowering the pH (5 m M MES/Tris) from 6.5 to 4.5 resulted in strong, long-lasting growth promotion. Surprisingly, increasing the pH from 6.5 to 8.5 also resulted in strong growth promotion. In LC roots acidification of the medium (pH 6.5 to 4.5) resulted in transient growth stimulation followed by a gradual decline in the growth rate toward zero. Exposure of LC roots to high pH (pH shift from 6.5 to 8.5) also promoted growth. Addition of EGTA resulted in strong growth promotion in both LC and HC roots. The ability of EGTA to stimulate growth appeared not to be related to H⁺ release from EGTA upon Ca²⁺ chelation since, 1) LC roots showed a strong and prolonged response to EGTA, but only a transient response to acid pH, and 2) promotion of growth by EGTA was observed in strongly buffered solutions. We also examined the pH dependence of the release of ⁴⁵Ca²⁺ from roots of 3-day-old seedlings grown from grains imbibed in ⁴⁵Ca²⁺. Release of ⁴⁵Ca²⁺ from the root into agar blocks placed on the root surface was greater the more acidic the pH of the blocks. The results indicate that Ca²⁺ may be necessary for the acid growth response in roots.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.