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Biomedical subjects

L. V. Kochian

Publications and source records attributed to L. V. Kochian.

15 recordsLinked to original sources

Induction of the Root Cell Plasma Membrane Ferric Reductase (An Exclusive Role for Fe and Cu).

Induction of ferric reductase activity in dicots and nongrass monocots is a well-recognized response to Fe deficiency. Recent evidence has shown that Cu deficiency also induces plasma membrane Fe reduction. In this study we investigated whether other nutrient deficiencies could also induce ferric reductase activity in roots of pea (Pisum sativum L. cv Sparkle) seedlings. Of the nutrient deficiencies tested (K, Mg, Ca, Mn, Zn, Fe, and Cu), only Cu and Fe deficiencies elicited a response. Cu deficiency induced an activity intermediate between Fe-deficient and control plant activities. To ascertain whether the same reductase is induced by Fe and Cu deficiency, concentration- and pH-dependent kinetics of root ferric reduction were compared in plants grown under control, -Fe, and -Cu conditions. Additionally, rhizosphere acidification, another process induced by Fe deficiency, was quantified in pea seedlings grown under the three regimes. Control, Fe-deficient, and Cu-deficient plants exhibited no major differences in pH optima or Km for the kinetics of ferric reduction. However, the Vmax for ferric reduction was dramatically influenced by plant nutrient status, increasing 16- to 38-fold under Fe deficiency and 1.5- to 4-fold under Cu deficiency, compared with that of control plants. These results are consistent with a model in which varying amounts of the same enzyme are deployed on the plasma membrane in response to plant Fe or Cu status. Rhizosphere acidification rates in the Cu-deficient plants were similarly intermediate between those of the control and Fe-deficient plants. These results suggest that Cu deficiency induces the same responses induced by Fe deficiency in peas.

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Al Inhibits Both Shoot Development and Root Growth in als3, an Al-Sensitive Arabidopsis Mutant.

In als3, an Al-sensitive Arabidopsis mutant, shoot development and root growth are sensitive to Al. Mutant als3 seedlings grown in an Al-containing medium exhibit severely inhibited leaf expansion and root growth. In the presence of Al, unexpanded leaves accumulate callose, an indicator of Al damage in roots. The possibility that the inhibition of shoot development in als3 is due to the hyperaccumulation of Al in this tissue was examined. However, it was found that the levels of Al that accumulated in shoots of als3 are not different from the wild type. The inhibition of shoot development in als3 is not a consequence of nonspecific damage to roots, because other metals (e.g. LaCl3 or CuSO4) that strongly inhibit root growth did not block shoot development in als3 seedlings. Al did not block leaf development in excised als3 shoots grown in an Al-containing medium, demonstrating that the Al-induced damage in als3 shoots was dependent on the presence of roots. This suggests that Al inhibition of als3 shoot development may be a delocalized response to Al-induced stresses in roots following Al exposure.

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Possible Involvement of Al-Induced Electrical Signals in Al Tolerance in Wheat.

The relationship between Al-induced depolarization of root-cell transmembrane electrical potentials (Em) and Al tolerance in wheat (Triticum aestivum L.) was investigated. Al exposure induced depolarizations of Em in the Al-tolerant wheat cultivars Atlas and ET3, but not in the Al-sensitive wheat cultivars Scout and ES3. The depolarizations of Em occured in root cap cells and as far back as 10 mm from the root tip. The depolarization was specific to Al3+; no depolarization was observed when roots were exposed to the rhizotoxic trivalent cation La3+. The Al-induced depolarization occurred in the presence of anion-channel antagonists that blocked the release of malate, indicating that the depolarization is not due to the electrogenic efflux of malate2-. K+-induced depolarizations in the root cap were of the same magnitude as Al-induced depolarizations, but did not trigger malate release, indicating that Al-induced depolarization of root cap cell membrane potentials is probably linked to, but is not sufficient to trigger, malate release.

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Aluminum Interactions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles Isolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars.

The role of Al interactions with root-cell plasma membrane (PM) Ca2+ channels in Al toxicity and resistance was studied. The experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Triticum aestivum L.) cultivars (Al-sensitive Scout 66 and Al-resistant Atlas 66). We previously used this technique to characterize a voltage-dependent Ca2+ channel in the wheat root PM (J.W. Huang, D.L. Grunes, L.V. Kochian [1994] Proc Natl Acad Sci USA 91: 3473-3477). We found that Al3+ effectively blocked this PM Ca2+ channel; however, Al3+ blocked this Ca2+ channel equally well in both the Al-sensitive and -resistant cultivars. It was found that the differential genotypic sensitivity of this Ca2+ transport system to Al in intact roots versus isolated PM vesicles was due to Al-induced malate exudation localized to the root apex in Al-resistant Atlas but not in Al-sensitive Scout. Because malate can effectively chelate Al3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to Al in intact roots of Al-resistant versus Al-sensitive wheat cultivars is probably due to the maintenance of lower Al3+ activities in the root apical rhizosphere of the resistant cultivar.

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Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea Using a Chelator Buffer System to Control Free Fe2+ in Solution.

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and non-specific binding of Fe to cell walls. In this study, a Fe(II)-3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4[prime]4"-disulfonic acid buffer system was used to control free Fe2+ in uptake solutions. Additionally, desorption methodologies were developed to adequately remove nonspecifically bound Fe from the root apoplasm. This enabled us to quantify unidirectional Fe2+ influx via radiotracer (59Fe) uptake in roots of pea (Pisum sativum cv Sparkle) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx into roots was dramatically inhibited by low temperature, indicating that the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were stimulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited higher Fe2+ influx rates than +Fe-grown Sparkle. These results suggest that either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.

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Multiple Aluminum-Resistance Mechanisms in Wheat (Roles of Root Apical Phosphate and Malate Exudation).

Although it is well known that aluminum (Al) resistance in wheat (Triticum aestivum) is multigenic, physiological evidence for multiple mechanisms of Al resistance has not yet been documented. The role of root apical phosphate and malate exudation in Al resistance was investigated in two wheat cultivars (Al-resistant Atlas and Al-sensitive Scout) and two near-isogenic lines (Al-resistant ET3 and Al-sensitive ES3). In Atlas Al resistance is multigenic, whereas in ET3 resistance is conditioned by the single Alt1 locus. Based on root- growth experiments, Atlas was found to be 3-fold more resistant in 20 [mu]M Al than ET3. Root-exudation experiments were conducted under sterile conditions; a large malate efflux localized to the root apex was observed only in Atlas and in ET3 and only in the presence of Al (5 and 20 [mu]M). Furthermore, the more Al-resistant Atlas exhibited a constitutive phosphate release localized to the root apex. As predicted from the formation constants for the Al-malate and Al-phosphate complexes, the addition of either ligand to the root bathing solution alleviated Al inhibition of root growth in Al-sensitive Scout. These results provide physiological evidence that Al resistance in Atlas is conditioned by at least two genes. In addition to the alt locus that controls Al-induced malate release from the root apex, other genetic loci appear to control constitutive phosphate release from the apex. We suggest that both exudation processes act in concert to enhance Al exclusion and Al resistance in Atlas.

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Physiological Characterization of Root Zn2+ Absorption and Translocation to Shoots in Zn Hyperaccumulator and Nonaccumulator Species of Thlaspi.

Radiotracer techniques were employed to characterize 65Zn2+ influx into the root symplasm and translocation to the shoot in Thlaspi caerulescens, a Zn hyperaccumulator, and Thlaspi arvense, a nonaccumulator. A protocol was developed that allowed us to quantify unidirectional 65Zn2+ influx across the root-cell plasma membrane (20 min of radioactive uptake followed by 15 min of desorption in a 100 [mu]M ZnCl2 + 5 mM CaCl2 solution). Concentration-dependent Zn2+ influx in both Thlaspi species yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The linear kinetic component was shown to be cell-wall-bound Zn2+ remaining in the root after desorption, and the saturable component was due to Zn2+ influx across the root-cell plasma membrane. This saturable component followed Michaelis-Menten kinetics, with similar apparent Michaelis constant values for T. caerulescens and T. arvense (8 and 6 [mu]M, respectively). However, the maximum initial velocity for Zn2+ influx in T. caerulescens root cells was 4.5-fold higher than for T. arvense, indicating that enhanced absorption into the root is one of the mechanisms involved in Zn hyperaccumulation. After 96 h 10-fold more 65Zn was translocated to the shoot of T. caerulescens compared with T. arvense. This indicates that transport sites other than entry into the root symplasm are also stimulated in T. caerulescens. We suggest that although increased root Zn2+ influx is a significant component, transport across the plasma membrane and tonoplast of leaf cells must also be critical sites for Zn hyperaccumulation in T. caerulescens.

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Aluminum Inhibition of the Inositol 1,4,5-Trisphosphate Signal Transduction Pathway in Wheat Roots: A Role in Aluminum Toxicity?

In crop plants, aluminum (Al) rhizotoxicity is a major problem worldwide; however, the cause of Al toxicity remains elusive. The effects of Al on the inositol 1,4,5-trisphosphate (Ins[1,4,5]P3)-mediated signal transduction pathway were investigated in wheat roots. Exogenously applied Al (50 [mu]M) rapidly inhibited root growth (<2 hr) but did not affect general root metabolism. An Ins(1,4,5)P3 transient was generated in root tips, either before or after exposure to Al for 1 hr, by treating the roots with H2O2 (10 mM). Background (unstimulated) levels of Ins(1,4,5)P3 were similar in both Al-treated and Al-untreated root apices. However, H2O2-stimulated levels of Ins(1,4,5)P3 in root apices showed a significant (>50%) reduction after Al exposure in comparison with untreated controls, indicating that Al may be interfering with the phosphoinositide signaling pathway. When phospholipase C (PLC) was assayed directly in the presence of Al or other metal cations in microsomal membranes, AlCl3 and Al-citrate specifically inhibited PLC action in a dose-dependent manner and at physiologically relevant Al levels. Al exposure had no effect on inositol trisphosphate dephosphorylation or on a range of enzymes isolated from wheat roots, suggesting that Al exposure may specifically target PLC. Possible mechanisms of PLC inhibition by Al and the role of Ins(1,4,5)P3 in Al toxicity and growth are discussed. This study provides compelling evidence that the phytotoxic metal cation Al has an intracellular target site that may be integrally involved in root growth.

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Ammonium Uptake by Rice Roots (III. Electrophysiology).

The transmembrane electrical potential differences ([delta][psi]) were measured in epidermal and cortical cells of intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings grown in 2 or 100 [mu]M NH4+ (G2 or G100 plants, respectively). In modified Johnson's nutrient solution containing no nitrogen, [delta][psi] was in the range of -120 to -140 mV. Introducing NH4+ to the bathing medium caused a rapid depolarization. At the steady state, average [delta][psi] of G2 and G100 plants were -116 and -89 mV, respectively. This depolarization exhibited a biphasic response to external NH4+ concentration similar to that reported for 13NH4+ influx isotherms (M.Y. Wang, M.Y. Siddiqi, T.J. Ruth, A.D.M. Glass [1993] Plant Physiol 103: 1259-1267). Plots of membrane depolarization versus 13NH4+ influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a breakpoint between two concentration ranges around 1 mM NH4+. The extent of depolarization was also influenced by nitrogen status, which was larger for G2 plants than for G100 plants. Depolarization of [delta][psi] due to NH4+ uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethylstilbestrol). The results of these observations are discussed in the context of the mechanisms of NH4+ uptake by high- and low-affinity transport systems operating across the plasma membranes of root cells.

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Aluminum Effects on Calcium (45Ca2+) Translocation in Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars (Differential Responses of the Root Apex versus Mature Root Regions).

The influence of Al exposure on long-distance Ca2+ translocation from specific root zones (root apex or mature root) to the shoot was studied in intact seedlings of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). Seedlings were grown in 100 [mu]M CaCl2 solution (pH 4.5) for 3 d. Subsequently, a divided chamber technique using 45Ca2+-labeled solutions (100 [mu]M CaCl2 with or without 5 or 20 [mu]M AlCl3, pH 4.5) was used to study Ca2+ translocation from either the terminal 5 to 10 mm of the root or a 10-mm region of intact root approximately 50 mm behind the root apex. The Al concentrations used, which were toxic to Scout 66, caused a significant inhibition of Ca2+ translocation from the apical region of Scout 66 roots. The same Al exposures had a much smaller effect on root apical Ca2+ translocation in Atlas 66. When a 10-mm region of the mature root was exposed to 45Ca2+, smaller genotypic differences in the Al effects effects on Ca2+ translocation were observed, because the degree of Al-induced inhibition of Ca2+ translocation was less than that at the root apex. Exposure of the root apex to Al inhibited root elongation by 70 to 99% in Scout 66 but had a lesser effect (less than 40% inhibition) in Atlas 66. When a mature root region was exposed to Al, root elongation was not significantly affected in either cultivar. These results demonstrate that genotypic differences in Al-induced inhibition of Ca2+ translocation and root growth are localized primarily in the root apex. The pattern of Ca2+ translocation within the intact root was mainly basipetal, with most of the absorbed Ca2+ translocated toward the shoot. A small amount of acropetal Ca2+ translocation from the mature root regions to the apex was also observed, which accounted for less than 5% of the total Ca2+ translocation within the entire root. Because Ca2+ translocation toward the root apex is limited, most of the Ca2+ needed for normal cellular function in the apex must be absorbed from the external solution. Thus, continuous Al disruption of Ca2+ absorption into cells of the root apex could alter Ca2+ nutrition and homeostasis in these cells and could play a pivotal role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars.

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Compartmentation Analysis of Paraquat Fluxes in Maize Roots as a Means of Estimating the Rate of Vacuolar Accumulation and Translocation to Shoots.

Efflux analysis conducted after five loading periods of various lengths (2, 6, 12, 18, or 24 h) was used to investigate uptake, compartmentation, and translocation of [14C]paraquat in maize (Zea mays L.) seedlings. The time course for net paraquat uptake (paraquat concentration in uptake solution = 25[mu]M) into maize roots was linear (56.7 nmol g-1 root fresh weight h-1) for 24 h. Estimates of changes in paraquat content in the vacuole, cytoplasm, and cell wall after 2-, 6-, 12-, 18-, and 24-h loading periods indicated that the cell wall saturated rapidly, whereas accumulation of paraquat into the vacuole increased linearly (12.4 nmol g-1 root fresh weight h-1) over 24 h. In contrast to vacuolar accumulation, cytoplasmic paraquat content appeared to approach saturation. The half-time for paraquat efflux from the cell wall (16.6 min [plus or minus] 1.2 SD) and cytoplasm (58.8 min [plus or minus] 8.9 SD remained relatively constant regardless of the length of the loading period, whereas the half-time for efflux from the vacuole was considerably longer and increased linearly with increased loading time (6.1-18.7 h). The time course for paraquat translocation to the shoot was linear within a 24-h exposure to radiolabeled herbicide, but translocation did not begin until 5 h after initiation of treatment. The experimental approach used in these experiments provides a valuable method for examining the movement of paraquat in maize seedlings. Results indicate that the herbicide slowly accumulates in the vacuole of root cells but is also translocated to the shoot.

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Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance.

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.

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Effects of Acetyl-Coenzyme A Carboxylase Inhibitors on Root Cell Transmembrane Electric Potentials in Graminicide-Tolerant and -Susceptible Corn (Zea mays L.).

Herbicidal activity of aryloxyphenoxypropionate and cyclohexanedione herbicides (graminicides) has been proposed to involve two mechanisms: inhibition of acetyl-coenzyme A carboxylase (ACCase) and depolarization of cell membrane potential. We examined the effect of aryloxyphenoxypropionates (diclofop and haloxyfop) and cyclohexanediones (sethoxydim and clethodim) on root cortical cell membrane potential of graminicide-susceptible and -tolerant corn (Zea mays L.) lines. The graminicide-tolerant corn line contained a herbicide-insensitive form of ACCase. The effect of the herbicides on membrane potential was similar in both corn lines. At a concentration of 50 [mu]M, the cyclohexanediones had little or no effect on the membrane potential of root cells. At pH 6, 50 [mu]M diclofop, but not haloxyfop, depolarized membrane potential, whereas both herbicides (50 [mu]M) dramatically depolarized membrane potential at pH 5. Repolarization of membrane potential after removal of haloxyfop and diclofop from the treatment solution was incomplete at pH 5. However, at pH 6 nearly complete repolarization of membrane potential occurred after removal of diclofop. In graminicide-susceptible corn, root growth was significantly inhibited by a 24-h exposure to 1 [mu]M haloxyfop or sethoxydim, but cell membrane potential was unaffected. In gramincide-tolerant corn, sethoxydim treatment (1 [mu]M, 48 h) had no effect on root growth, whereas haloxyfop (1 [mu]M, 48 h) inhibited root growth by 78%. However, membrane potential was the same in roots treated with 1 [mu]M haloxyfop or sethoxydim. The results of this study indicate that graminicide tolerance in the corn line used in this investigation is not related to an altered response at the cell membrane level as has been demonstrated with other resistant species.

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Characterization of Paraquat Transport in Protoplasts from Maize (Zea mays L.) Suspension Cells.

Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat. [14C]Paraquat uptake was measured in 400-[mu]L centrifuge tubes using silicon oil centrifugation techniques. Approximately 50% of accumulation from a 100 [mu]M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min. Membrane binding accounted for about 30% of apparent accumulation. Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component. The Km of the saturable component was 132 [mu]M, and the Vmax was 0.512 nmol [mu]L of protoplasts-1 min-1. In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent. Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response. This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface. Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive. Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake. These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings.

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