Polar Transport of Auxin across Gravistimulated Roots of Maize and Its Enhancement by Calcium

The effect of Ca on the polar movement of [³H]indoleacetic acid ([³H] IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [³H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl₂ into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl₂ was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [³H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl₂. With the inclusion of CaCl₂ in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [³H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.     

Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots

The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl₂ so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl₂, plus 100 millimolar NaCl plus 10 millimolar CaCl₂. The hydraulic conductivities (Lp_r) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp_r for the different treatments were 2.8, 1.7, 2.8, and 3.4·10⁻⁷ meters per second per megapascals and the endosmotic hydrostatic Lp_r were 2.4, 1.5, 2.7, and 2.3·10⁻⁷ meters per second per megapascals, respectively. Exosmotic Lp_r of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60·10⁻⁷ meters per second per megapascals and the endosmotic Lp_r were 0.53, 0.21, 0.56, and 0.54·10⁻⁷ meters per second per megapascals, respectively. The osmotic Lp_r was significantly smaller (4-5 times) than hydrostatic Lp_r. However, both hydrostatic and osmotic Lp_r experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp_r significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp_r. The low Lp_r values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5·10⁻⁹ meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects.     

Incorporation of [C]Glucose into Cell Wall Polysaccharides of Cotton Roots: Effects of NaCl and CaCl(2)

Cotton (Gossypium hirsutum L. cv Acala SJ-2) seedlings were grown in nutrient solutions with four combinations of NaCl (0.1 and 150 millimolar) and CaCl₂ (1 and 10 millimolar) for 7 days, and then exposed to [¹⁴C]glucose for 5 hours. Uptake and incorporation of [¹⁴C]glucose into various cell wall fractions of the root tips were determined. At 1 millimolar Ca²⁺, treatment with 150 millimolar NaCl slightly stimulated uptake but considerably inhibited glucose incorporation into noncellulosic and cellulosic polysaccharides. Supplemental Ca²⁺ did not affect incorporation of glucose into the noncellulosic fraction (regardless of NaCl treatment) but completely alleviated the inhibitory effect of NaCl on glucose incorporation into cellulose. We suggest that high Na⁺ concentrations reduce synthesis of cellulose in cotton roots via disturbance of plasma membrane integrity and that supplemental Ca²⁺ counteracts this effect. The effects on cellulose biosynthesis are proposed to be related to Ca²⁺ displacement from the plasma membrane.     

Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue

Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of ¹⁴C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.

Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl₂. Unloading was stimulated by 15 millimolar ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid which partially reversed the inhibitory effects of Ca²⁺. Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses.

     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Effects of NaCl and CaCl(2) on Water Transport across Root Cells of Maize (Zea mays L.) Seedlings

The effect of salinity and calcium levels on water flows and on hydraulic parameters of individual cortical cells of excised roots of young maize (Zea mays L. cv Halamish) plants have been measured using the cell pressure probe. Maize seedlings were grown in one-third strength Hoagland solution modified by additions of NaCl and/or extra calcium so that the seedlings received one of four treatments: control; +100 millimolar NaCl; +10 millimolar CaCl₂; +100 millimolar NaCl + 10 millimolar CaCl₂. From the hydrostatic and osmotic relaxations of turgor, the hydraulic conductivity (Lp) and the reflection coefficient (σ_s) of cortical cells of different root layers were determined. Mean Lp values in the different layers (first to third, fourth to sixth, seventh to ninth) of the four different treatments ranged from 11.8 to 14.5 (Control), 2.5 to 3.8 (+NaCl), 6.9 to 8.7 (+CaCl₂), and 6.6 to 7.2 · 10⁻⁷ meter per second per megapascal (+NaCl + CaCl₂). These results indicate that salinization of the growth media at regular calcium levels (0.5 millimolar) decreased Lp significantly (three to six times). The addition of extra calcium (10 millimolar) to the salinized media produced compensating effects. Mean cell σ_s values of NaCl ranged from 1.08 to 1.16, 1.15 to 1.22, 0.94 to 1.00, and 1.32 to 1.46 in different root cell layers of the four different treatments, respectively. Some of these σ_s values were probably overestimated due to an underestimation of the elastic modulus of cells, σ_s values of close to unity were in line with the fact that root cell membranes were practically not permeable to NaCl. However, the root cylinder exhibited some permeability to NaCl as was demonstrated by the root pressure probe measurements that resulted in σ_sr of less than unity. Compared with the controls, salinity and calcium increased the root cell diameter. Salinized seedlings grown at regular calcium levels resulted in shorter cell length compared with control (by a factor of 2). The results demonstrate that NaCl has adverse effects on water transport parameters of root cells. Extra calcium could, in part, compensate for these effects. The data suggest a considerable apoplasmic water flow in the root cortex. However, the cell-to-cell path also contributed to the overall water transport in maize roots and appeared to be responsible for the decrease in root hydraulic conductivity reported earlier (Azaizeh H, Steudle E [1991] Plant Physiol 97: 1136-1145). Accordingly, the effect of high salinity on the cell Lp was much larger than that on root Lp_r.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Potassium transport in corn roots : I. Resolution of kinetics into a saturable and linear component

Influx isotherms were obtained for ⁸⁶Rb⁺ uptake into 2-cm corn (Zea mays [A632 × (C3640 × Oh43)] root segments for both low- (0.2 millimolar CaSO₄) and high-salt (0.2 millimolar CaSO₄ + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K⁺ influx, our isotherms were smooth, nonsaturating curves that approached linearity at K⁺ (Rb⁺) concentrations above 1 millimolar. The kinetics for K⁺ transport could be resolved into saturable and linear components. The saturable components yielded K_m values of 16 and 86 micromolar for low- and high-salt roots, respectively, while V_max values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K⁺ transport.

Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor.

The effects of NEM and PCMBS on K⁺ efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished.

The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion.

     

ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles

An anion-sensitive ATP-dependent H⁺ transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H⁺ transport observed: strict dependence on Cl⁻; inhibition by SO₄²⁻ and NO₃⁻; insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl₂ (H⁺/Ca²⁺ antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl₂ with a K_m value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H⁺ transport significantly. However, ADP was a potent competitive inhibitor with a K_i value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.     

Calcium Dependence of Rapid Auxin Action in Maize Roots

We investigated the interaction of Ca²⁺ and auxin on root elongation in seedlings of Zea mays L. The seedlings were raised either in the presence of Ca²⁺ (high calcium; HC = imbibed and raised in 10 millimolar CaCl₂), in the absence of additional Ca²⁺ (intermediate calcium; IC = imbibed and raised in distilled H₂O, calcium supply from seed only), or without additional Ca²⁺ and subsequently depleting them of Ca²⁺ (low calcium; LC = imbibed and raised in distilled H₂O and subsequently treated with 1 millimolar ethyleneglycol-bis-[β-aminoethylether]-N,N,N′,N′ -tetraacetic acid [EGTA]). Exposure of roots of either HC or IC seedlings to auxin concentrations from 0.1 to 10 micromolar resulted in strong inhibition of elongation. In roots of LC seedlings, on the other hand, auxin concentrations as high as 10 micromolar caused only slight inhibition of elongation. Adding 0.5 millimolar Ca²⁺ to LC roots in the presence of IAA allowed normal expression of the inhibitory action of the hormone. Inhibition of elongation in IC roots by indoleacetic acid was reversible upon treatment of the roots with 1 millimolar EGTA. The inhibitory action of auxin could then be re-established by supplying 0.5 millimolar Ca²⁺. The data indicate that Ca²⁺ may be necessary to the growth-regulating action of auxin. The significance of this finding is discussed with respect to the potential role of Ca²⁺ as a second messenger of auxin action and the relevance of this model to recent evidence for gravi-induced redistribution of Ca²⁺ and its role in establishing gravitropic curvature.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Effects of ouabain and low temperature on the sodium efflux pump in excised corn roots

Ouabain (0.05 millimolar) and low temperature (4 C) both caused the tissue Na⁺ content of excised 5-day-old corn roots to increase, indicating that there is an inhibition of the Na⁺ efflux pump. Na⁺ efflux was measured utilizing three different methods. Each method gave similar results in terms of rate and ouabain sensitivity. With one of these methods, the compartmental efflux method, it was demonstrated that rates for Na⁺ efflux increase as the external Na⁺ concentration is increased; e.g. the efflux rates are 0.529, 1.78, and 3.64 microequivalents per gram fresh weight per hour for external NaCl concentrations of 1, 10, and 30 millimolar, respectively. The data indicate that the Na⁺ efflux pump is located in the plasmalemma of root cells.     

Isolation and transport properties of protoplasts from cortical cells of corn roots

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl₂, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 10³/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.

Cortical cell protoplasts exhibited energy-dependent uptake of K⁺ (⁸⁶Rb), H₂³²PO₄⁻, and ³⁶Cl⁻ as well as net H⁺ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K⁺ was highest between pH 7.5 and 8.0, whereas the influx of H₂PO₄⁻ was greatest between pH 4.0 and 5.0. K⁺ and H₂PO₄⁻ influx and net H⁺ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl⁻ was low, but not greatly different from that observed for other plant cells. K⁺ flux was somewhat high, probably because the K⁺ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.

     

Cadmium alteration of root physiology and potassium ion fluxes

Segments of oat (Avena sativa L.) roots which had been exposed to 1 millimolar CdSO₄ in quarter-strength Hoagland No. 1 solution exhibited decreased respiratory rates, ATP levels, membrane-bound ATPase activity, and reduced K⁺ fluxes. Respiration and ATP levels were decreased after a 2-hour treatment with 1 millimolar CdSO₄ to 65 and 75%, respectively, of control rates. A membrane-bound, Mg²⁺-dependent, K⁺-stimulated acid ATPase was rapidly inhibited to 12% of control activity in the presence of 1 millimolar CdSO₄. Potassium uptake into root segments was inhibited to 80% of control values after 30 minutes in the presence of CdSO₄. A 2-hour pretreatment of root segments with CdSO₄ inhibited K⁺ uptake to 15% of control values. Cytoplasmic K⁺ efflux was inhibited with 1 millimolar CdSO₄.

The rates and the degree of Cd²⁺ inhibition of the parameters listed above suggest that one of the first sites of Cd²⁺ action is the plasmalemma K⁺ carrier (ATPase) in oat roots.

     

Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells : Its Simulation in Extracellular KCl and Prevention by Calcium

Freezing injury, in onion bulb tissue, is known to cause enhanced K⁺ efflux accompanied by a small but significant loss of Ca²⁺ following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K⁺ into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCl. These symptoms were prevented by adding 20 millimolar CaCl₂ to the extracellular KCl solution. Our results provide evidence that loss of cellular Ca²⁺ plays an important role in the initiation and the progression of freezing injury.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

Role of Ca and EGTA on Stomatal Movements in Commelina communis L

Ca²⁺ (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(β-aminoethyl ether) N,N′tetraacetic acid (EGTA) (2 millimolar), a Ca²⁺ chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl₂. The results demonstrate the importance of Ca²⁺ in the regulation of stomatal closure and point to a role of Ca²⁺ in the regulation of K⁺ efflux from stomatal guard cells.     

Isolation of amyloplasts from developing maize endosperm

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4°C or 5 hours at 30°C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl₂. After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.