Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeño and South American 5) maize genotypes. In a growth solution containing 6 M Al³⁺, Tuxpeño and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al³⁺ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.Abbreviations SA3 South American 3, an Al-tolerant maize cultivar - SA5 South American 5, an Al-sensitive maize cultivar The authors would like to express their appreciation to Drs. John Thompson, Ross Welch and Mr. Stephen Schaefer for their training and guidance in the use of the chromatography systems. This work was supported by a Swiss National Science Foundation Fellowship to Didier Pellet, and U.S. Department of Agriculture/National Research Initiative Competitive Grant 93-37100-8874 to Leon Kochian. We would also like to thank Drs. S. Pandey and E. Ceballos from the CIMMYT Regional office at CIAT Cali, Colombia for providing seed for the maize varieties and inbred line.     

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.     

Nitrogen and potassium fertilization and environmental factors affecting the grass tetany hazard of wheat forage

Summary The effects of temperature and soil moisture levels on the chemical composition of wheat forage grown in growth chambers were studied. In addition to the environmental variables, K and N fertilization effects were studied. In all the studies, increasing levels of K fertilization depressed the Mg and Ca concentration of the shoots. Nitrogen fertilization increased the Mg concentration but had no effect on the Ca concentration of the plants. N fertilization depressed the K concentration in the soil moisture experiment, but had no effect on K concentration in the temperature experiment. Increasing the temperature from 10 to 20°C did not affect the Mg and Ca concentration of the shoots, but the K concentration declined due to dilution effects caused by the greater yield at the higher temperature. In the soil moisture level experiment the K, Mg and Ca concentration in wheat tended to decline with soil moisture level due to dilution effects. Calculations showed that uptake of K was regulated primarily by diffusion of K from the soil to the plant root and that the uptake of Mg was regulated by the uptake process of the plant root and not by the nutrient transport process through the soil.This study was part of the program of the Center for Root-Soil Research. Dept. of Agronomy paper #1532.     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Aluminium and calcium transport interactions in intact roots and root plasmalemma vesicles from aluminium-sensitive and tolerant wheat cultivars

Recent research from our laboratory indicates that aluminium (Al) and calcium (Ca) transport interactions may play an important role in the mechanisms of Al phytotoxicity. In this study, we investigated the effects of Al on Ca²⁺ transport in intact roots of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). We used both a vibrating Ca²⁺-microelectrode technique and ⁴⁵Ca²⁺ to monitor Ca²⁺ influx in intact roots. Root apical Ca²⁺ uptake was immediately inhibited, when roots were exposed to Al levels that ultimately decreased root growth in Al-sensitive Scout 66. The Al-tolerant cultivar was able to resist this Al inhibition of Ca²⁺ uptake, and to resist Al inhibition of ⁴⁵Ca²⁺ translocation from roots to shoots. We also studied Ca²⁺ transport in right-side out plasmalemma vesicles isolated from roots of Al-sensitive and tolerant wheat cultivars. Calcium influx into the vesicles was mediated by a voltage-gated Ca²⁺ channel. Aluminium blocks the Ca²⁺ channel equally well in the plasmalemma vesicles isolated from Al-sensitive and Al-tolerant wheat roots. The results indicate that the differential response observed in intact roots is not due to differences in Ca²⁺ channels. The Al-tolerant wheat cultivar may have an ability to reduce Al³⁺ activity in the rhizosphere, thus reducing the Al-inhibition of Ca²⁺ influx.     

Involvement of multiple aluminium exclusion mechanisms in aluminium tolerance in wheat

The goal of this study was to determine if Al-chelators other than malate are released from root apices and are involved in Al-tolerance in different wheat (Triticum aestivum L.) genotypes. Also we wanted to establish if root exudates contribute to increases in rhizosphere pH around the root tip. In seedlings of Al-tolerant Atlas, we have documented a constitutive phosphate exudation from the root apex. Because phosphate can complex Al and bind protons, it could play an important role in Al tolerance, both via complexation of Al³⁺ and by contributing to the alkalinization of rhizosphere pH observed at the apex of Atlas. This study suggests that in wheat, Al-tolerance can be mediated by multiple exclusion mechanisms controlled by different genes.     

Aluminum effects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars

The role of Ca²⁺ transport in the mechanism of Al toxicity was investigated, using a Ca²⁺-selective microelectrode system to study Al effects on root apical Ca²⁺ fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl₂, pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca²⁺ uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl₃) immediately and dramatically inhibited Ca²⁺ uptake in Scout, whereas Ca²⁺ transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca²⁺ uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca²⁺ influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca²⁺ transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca²⁺ channels could play a role in the cellular mechanism of Al toxicity in higher plants.     

Effect of N,K, and P fertilization,N source,and clipping on potential tetany hazard of bromegrass

Summary The objective of this field study was to determine early-season effects of N source, N, K, and P fertilization, and clipping (to simulate grazing) on potential tetany hazard of bromegrass (Bromus inermis L.) as indicated by the chemical composition of its forage. Tetany is a metabolic disorder of ruminants resulting from forage with low Mg availability. Chemical components considered in the forage were inorganic cations, organic acids, aconitate, and per cent total N/per cent total water soluble carbohydrate (N/TWSC). Differences between the sum (in meq/kg) of inorganic cations (Mg, Ca, K, and Na) and inorganic anions (Cl, NO₃, H₂PO₄, and SO₄) in forage were defined as the concentration of organic acids (C-A). Soil was Parshall fsl, a Pachic Haploboroll. Yields and chemical composition of oven-dried forage from previously unclipped and reclipped plots were determined at 3-week intervals beginning May 22 and June 12, respectively. A water budget was determined using soil-water and rainfall data.Forage yields were increased 2- to 3-fold by N fertilization with the NO₃-N source generally outyielding the NH₄-N source. A slight additional yield response to that obtained with N alone was obtained with K+P fertilization but not with K or P alone with or without N. Much less total forage was removed from reclipped plots than from unclipped plots. Forage Mg content was decreased only slightly by K or NH₄-N fertilization. Soil analysis indicated that high NH₄-N levels were present at the May 22 harvest. Magnesium and Ca concentrations were only slightly affected by N fertilization; however, K, K/(Ca+Mg), total N, C-A, and aconitate were increased. Reclipping increased Mg, N, K, N/TWSC, C-A, and aconitate. Estimates of blood-plasma Mg concentrations were obtained by using the data for plant N, K, and Mg. These estimates did not indicate increased tetany hazard as a result of reclipping, but did indicate increased tetany hazard from N fertilization. Forage C-A and aconitate concentrations were decreased by fertilization with KCl which seemed to have been caused by the increased Cl concentrations in the forage. Estimates of quantities of Mg, arriving at the root surfaces from the soil by mass flow, far exceeded amounts of Mg in the forage. Mass flow seemed to be the principal mechanism by which Mg and Ca arrived at root surfaces but this mechanism was much less important for K.This study indicated an increased potential tetany hazard resulting primarily from N fertilization with either NH₄-N or NO₃-N sources. However, the potential for increased forage and livestock-carrying capacity with N fertilization is very large. Therefore, management practices corroborated by livestock data are vitally needed to minimize tetany hazard while increasing bromegrass yields by N fertilization.Contribution from Soil, Water, and Air Sciences, North Central and Northeastern Regions, ARS-USDA.Follett, Power, and Grunes are soil scientists and Kleinis a biological laboratory technician. Follett is now National Program Staff Scientist, ARS, BARC-West, Beltsville, MD 20705. Power and Klein are at the USDA Northern Great Plains Research Center, Mandan, ND 58554, as formerly was Follett. Grunes is at the U.S. Plant, Soil and Nutrition Laboratory, Ithaca, NY 14853.