Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.)

To identify the chemical forms of aluminum (Al) transported from roots to shoots of tea plants (C. sinensis L.), ²⁷Al-nuclear magnetic resonance and ¹⁹F NMR spectroscopy were used to analyze xylem sap.The concentration of Al in collected xylem sap was 0.29 mM, twice as high as that of F. Catechins were not detected in xylem sap. The concentration of malic acid in xylem sap was higher than that of citric acid, whereas the concentration of oxalic acid was negligible.There were two signals in the ²⁷Al NMR spectra of xylem sap, a larger signal at 11 ppm and a smaller one at −1.5 ppm. The former signal was consistent with the peak for an Al-citrate model solution, suggesting that an Al-citrate complex was present in xylem sap. Although the latter signal at −1.5 ppm was thought to indicate the presence of an Al-F complex (at 1.7 ppm) in xylem sap, there was only one signal at −122 ppm in the ¹⁹F NMR spectrum of xylem sap, indicating that the main F complex in xylem sap was F⁻.These results indicate that Al might be translocated as a complex with citrate, while Al-malate, Al-oxalate and Al-F complexes are not major Al complexes in xylem sap of tea plants.     

High Aluminum Resistance in Buckwheat : II. Oxalic Acid Detoxifies Aluminum Internally

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi), which shows high Al resistance, accumulates Al in the leaves. The internal detoxification mechanism was studied by purifying and identifying Al complexes in the leaves and roots. About 90% of Al accumulated in the leaves was found in the cell sap, in which the dominant organic acid was oxalic acid. Purification of the Al complex in the cell sap of leaves by molecular-sieve chromatography resulted in a complex with a ratio of Al to oxalic acid of 1:3. A ¹³C-nuclear magnetic resonance study of the purified cell sap revealed only one signal at a chemical shift 164.4 ppm, which was assigned to the Al-chelated carboxylic group of oxalic acid. A ²⁷Al-nuclear magnetic resonance analysis revealed one major signal at the chemical shift of 16.0 to 17.0 ppm, with a minor signal at the chemical shift of 11.0 to 12 ppm in both the intact roots and their cell sap, which is consistent with the Al-oxalate complexes at 1:3 and 1:2 ratios, respectively. The purified cell sap was not phytotoxic to root elongation in corn (Zea mays). All of these results indicate that Al tolerance in the roots and leaves of buckwheat is achieved by the formation of a nonphytotoxic Al-oxalate (1:3) complex.     

Form of aluminium for uptake and translocation in buckwheat (Fagopyrum esculentum Moench)

 The forms of Al for uptake by the roots and translocation from the root to the shoot were investigated in a buckwheat (Fagopyrum esculentum Moench, cv. Jianxi) that accumulates Al in its leaves. The Al concentration in the xylem sap was 15-fold higher in the plants exposed to AlCl₃ than in those exposed to an Al-oxalate (1:3) complex, suggesting that the roots take up Al in the ionic form. The Al concentration in the xylem sap was 4-fold higher than that in the external solution after a 1-h exposure to AlCl₃ solution and 10-fold higher after a 2-h exposure. The Al concentration in the xylem sap increased with increasing Al concentration in the external solution. The Al uptake was not affected by a respiratory inhibitor, hydroxylamine, but significantly inhibited by the addition of La. These results suggest that Al uptake by the root is a passive process, and La³⁺ competes for the binding sites for Al³⁺ on the plasma membrane. The form of Al in the xylem sap was identified by ²⁷Al-nuclear magnetic resonance analysis. The chemical shift of ²⁷Al in the xylem sap was around 10.9 ppm, which is consistent with that of the Al-citrate complex. Furthermore, the dominant organic acid in the xylem sap was citric acid, indicating that Al was translocated in the form of Al-citrate complex. Because Al is present as Al-oxalate (1:3) in the root, the present data show that ligand exchange from oxalate to citrate occurs before Al is released to xylem. Received: 10 December 1999 / Accepted: 3 February 2000     

Distribution and chemical speciation of aluminum in the Al accumulator plant,Melastoma malabathricum L.

The Al accumulation mechanisms in an Al accumulator plant, Melastoma malabathricum L. (Melastoma), was investigated. Al was located in the upper epidermal cells and also distributed in mesophyll cells in leaf sections. In root sections, Al was found in all the root tissues, particularly in the epidermis and endodermis. Al concentrations in young leaves, mature leaves, old leaves, and roots were 8.0, 9.2, 14.4, and 10.1 mg g¹, respectively. Approximately 45% of total Al in oldest leaves, and approximately 60% of total Al in leaves of other positions and roots were extracted in Tris-HCl buffer (pH 7.0). Since Al in the residual parts was mostly dissolved in hot 0.5 M H₂SO₄ containing 2% cetyl trimethylammonium bromide, residual Al seemed to consist mainly of monomeric Al and Al bound to pectic substances and hemicellulose. Al in the Tris-HCl extract consisted of non-monomeric Al (complexed form). Oxalate concentration in the Tris-HCl extract in leaves was significantly higher in the +Al treatment than in the –Al treatment and there was a positive correlation between the Al concentration and oxalate concentration. ²⁷Al NMR spectrum of fresh leaves indicated the presence in the order of monomeric Al, Al-oxalate, Al-(oxalate)₂, and Al-(oxalate)₃ in intact leaves.     

Role of aluminum-binding ligands in aluminum resistance of Eucalyptus camaldulensis and Melaleuca cajuputi

We investigated the roles of Al-binding ligands in Al exclusion from roots and in internal Al detoxification in roots as Al resistance mechanisms in two Al-resistant Myrtaceae trees, Eucalyptus camaldulensis Dehnh. and Melaleuca cajuputi Powell. The amounts of ligands secreted from roots and contained in root tips of these species were compared with those of an Al-sensitive species, Melaleuca bracteata F. Muell., after the roots were exposed to 0 or 1 mM AlCl₃ solution. Secretion of well-known ligands (citrate, oxalate, and malate) from roots under Al treatment was low in all species. However, in E. camaldulensis, the Al-binding capacity of root exudates under Al treatment was considerable and was higher than that in M. bracteata. Gel filtration chromatography revealed that a low-molecular-weight Al-binding ligand was secreted from roots in response to Al only in E. camaldulensis. On the other hand, the Al-binding capacity of cell sap in root tips under Al treatment was similar for the resistant and sensitive species. These results suggest that Al exclusion by secretion of the unknown low-molecular-weight Al-binding ligand from roots contributes to the Al resistance of E. camaldulensis, whereas M. cajuputi has developed Al-resistance mechanisms other than secretion of ligands from roots or concentration of internal ligands in root tips.     

Form of Al changes with Al concentration in leaves of buckwheat

Buckwheat (Fagopyrum esculentum Moench. cv. Jianxi) is known as an Al-accumulating plant. The process leading to the accumulation of Al in the leaves was investigated, focusing on the chemical form of Al using 27Al-nuclear magnetic resonance. Leaves with different Al concentrations were prepared by growing buckwheat on a very acidic soil (Andosol) amended with or without CaCO3 (1 or 3 g x kg-1 soil). When the Al concentration of the leaves was lower, only one major signal was observed at a chemical shift of 16.1 ppm, which was assigned to an Al-oxalate complex at a 1:3 ratio. However, when the Al concentration of the leaves increased to a high level (e.g. 12 g Al kg-1), an additional signal at a chemical shift of 11.2 ppm was observed. This signal was assigned to an Al-citrate complex at a 1:1 ratio. In the leaf with a high Al concentration, both Al-oxalate (1:3) and Al-citrate (1:1) were detected in marginal and middle parts, while only Al-oxalate was detected in the basal part. The oxalate concentration did not differ very much between leaves with low and high Al concentrations at the same position, while citrate concentration significantly increased with increasing Al concentration when the oxalate/Al ratio became lower than 3.0. As the Al-citrate complex has been demonstrated to be the form of transport in the xylem, the results suggest that when internal oxalate is enough to form a complex with Al at a 3:1 ratio in the leaves with a low Al concentration, Al-citrate converts to Al-oxalate. However, this conversion does not occur in the leaves with a very high Al concentration, resulting in the coexistence of both Al-oxalate and Al-citrate complexes.     

Aluminum resistance in two cultivars of Zea mays L.: Root exudation of organic acids and influence of phosphorus nutrition

Root exudation of organic acids as Al-chelating compounds and P nutrition have been suggested to play a major role in Al-resistance in higher plants. Effects of Al exposure on maize plant growth, and organic acid root content and root exudation under various levels of P nutrition were examined. Sikuani, a Colombian maize cultivar tolerant to acid soils with high Al saturation, and Corso, a Swiss cultivar, were grown in sterile hydroponic conditions for 21 days. Al-caused inhibition of root growth was lower in Sikuani than in Corso. Al effect on plant growth was decreased with increasing P content in roots. Al content in roots increased with increasing P content and was higher in Sikuani than in Corso. When exposed to Al, the contents in root apices as well as the root exudation of citric and malic acids in Corso and citric, malic and succinic acids in Sikuani increased, and were higher in Sikuani than in Corso. Increased PEP carboxylase (PEPC) activity in root apices after Al exposure partially explained the variations of organic acid content in the roots. These Al-induced changes in PEPC activity, organic acid content and exudation were reduced in plants supplied with higher P concentrations during the 21 days prior to treatment. Increased secretion of organic acids after exposure to Al appeared to be specific to Al and was not totally explained by increased root content in organic acids.     

Role of organic acids in detoxification of aluminum in higher plants

Phytotoxicity of aluminum ion (Al3+) is a serious problem limiting crop production on acid soils. Organic acids with Al-chelating ability play an important role in the detoxification of Al both externally and internally. Al is detoxified externally by the secretion of organic acids such as citric, oxalic, and/or malic acids from the roots. The secretion of organic acids is highly specific to Al and the site of secretion is localized to the root apex. The kind of organic acids secreted as well as secretion pattern differ among plant species. There are two patterns of Al-induced secretion of organic acids: In pattern I, there is no discernible delay between the addition of Al and the onset of the release of organic acids. Activation of the anion channel seems to be involved in this pattern; In pattern II, there is a marked lag phase between the addition of Al and the onset of organic acid release. The action of genes related to the metabolism and secretion of organic acids seems to be involved in this pattern. Internal detoxification of Al in Al-accumulating plants is achieved by the formation of Al-organic acid complex. For instance, the complex of Al-citrate (1:1) in hydrangea and Al-oxalate (1:3) in buckwheat has been identified.     

The role of organic acids in the short- and long-term aluminum tolerance in maize seedlings (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Aluminum (Al) affects numerous physiological processes in plants. However, Al tolerance mechanisms mediated by increased synthesis of organic acids (OAs) have been outlined recently. In this study, we examined the role of OAs in the short (1–8 h) and long-term (4 days) Al tolerance in maize seedlings. Exposure to Al stress for 4 days results in a rapid inhibition of root growth. Al induced morphological changes in the maize roots, especially at a higher solution of Al concentration (1,000 μM Al). The increase in Al accumulation in roots, including strongly elevated levels of Al accumulated in root cell walls suggests that Al tolerance in maize is mediated in part by higher accumulation of Al in the roots. The enhanced citrate exudation, which was only observed at 1,000 μM Al may lead to detoxification of Al by formation of OA–Al complexes in the root apoplast. This mechanism has been suggested to play a significant role in Al resistance response in maize. The short-term responses underlying internal detoxification via OA-chelators were also investigated. Succinate, malate, citrate and total root OA contents decreased markedly, 2 h after the Al exposure. At 4 and 8 h time points, OA contents increased or remained unchanged, except for that of malate which decreased. The level of OAs in shoots, on the other hand, showed alterations that were less pronounced in response to Al. Specifically, the citrate and total OA concentrations significantly increased at 4 h, but showed a pronounced decrease at the 8 h time point. Based on our findings, we propose that multiple responses, including Al exclusion by Al accumulation in root cells and citrate efflux, may contribute towards higher Al resistance in maize. The rapid OA changes in responses to short-term Al treatment may not be responsible for Al tolerance. However, increased OA synthesis observed in this study may be involved in diminishing the stress triggered by Al. The molecular aspects underlying Al resistance mechanism via Al-induced expression of the enzymes catalyzing OA synthesis and metabolism remain to be elucidated.     

Aluminium alleviates manganese toxicity to rice by decreasing root symplastic Mn uptake and reducing availability to shoots of Mn stored in roots

Background and Aims Manganese (Mn) and aluminium (Al) phytotoxicities occur mainly in acid soils. In some plant species, Al alleviates Mn toxicity, but the mechanisms underlying this effect are obscure.Methods Rice (Oryza sativa) seedlings (11 d old) were grown in nutrient solution containing different concentrations of Mn²⁺ and Al³⁺ in short-term (24 h) and long-term (3 weeks) treatments. Measurements were taken of root symplastic sap, root Mn plaques, cell membrane electrical surface potential and Mn activity, root morphology and plant growth.Key Results In the 3-week treatment, addition of Al resulted in increased root and shoot dry weight for plants under toxic levels of Mn. This was associated with decreased Mn concentration in the shoots and increased Mn concentration in the roots. In the 24-h treatment, addition of Al resulted in decreased Mn accumulation in the root symplasts and in the shoots. This was attributed to higher cell membrane surface electrical potential and lower Mn²⁺ activity at the cell membrane surface. The increased Mn accumulation in roots from the 3-week treatment was attributed to the formation of Mn plaques, which were probably related to the Al-induced increase in root aerenchyma.Conclusions The results show that Al alleviated Mn toxicity in rice, and this could be attributed to decreased shoot Mn accumulation resulting from an Al-induced decrease in root symplastic Mn uptake. The decrease in root symplastic Mn uptake resulted from an Al-induced change in cell membrane potential. In addition, Al increased Mn plaques in the roots and changed the binding properties of the cell wall, resulting in accumulation of non-available Mn in roots.     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.     

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H₂O₂ generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H₂O₂. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.     

Elevated oxalate oxidase activity is correlated with Al-induced plasma membrane injury and root growth inhibition in young barley roots

In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several (3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting its role in oxidative stress and subsequent cell death caused by Al toxicity in plants.     

Aluminium is essential for root growth and development of tea plants (Camellia sinensis)

On acid soils, the trivalent aluminium ion (Al³⁺) predominates and is very rhizotoxic to most plant species. For some native plant species adapted to acid soils including tea (Camellia sinensis), Al³⁺ has been regarded as a beneficial mineral element. In this study, we discovered that Al³⁺ is actually essential for tea root growth and development in all the tested varieties. Aluminum ion promoted new root growth in five representative tea varieties with dose‐dependent responses to Al³⁺ availability. In the absence of Al³⁺, the tea plants failed to generate new roots, and the root tips were damaged within 1 d of Al deprivation. Structural analysis of root tips demonstrated that Al was required for root meristem development and activity. In situ morin staining of Al³⁺ in roots revealed that Al mainly localized to nuclei in root meristem cells, but then gradually moved to the cytosol when Al³⁺ was subsequently withdrawn. This movement of Al³⁺ from nuclei to cytosols was accompanied by exacerbated DNA damage, which suggests that the nuclear‐targeted Al primarily acts to maintain DNA integrity. Taken together, these results provide novel evidence that Al³⁺ is essential for root growth in tea plants through maintenance of DNA integrity in meristematic cells.     

The formation of iron plaques on roots and rhizomes of the seagrass Cymodocea serrulata (R. Brown) Ascherson with implications for sulphide intrusion

Despite the fact that iron plaque formation is ubiquitous in aquatic macrophytes and has been known for several decades, there are few reports of plaque occurrence in seagrasses to date. Herein we present the first microscopical observation and chemical quantification of iron (Fe) plaques on the shoots, rhizomes and roots of the seagrass Cymodocea serrulata (R. Brown) Ascherson collected from intertidal seagrass beds in Thailand. Plaques were observed on shoot bases, rhizomes and roots with the highest concentrations of iron in the plaques from the roots, reaching an average of 509 μmol gDW⁻¹. Interestingly, the most negative stable sulphur isotope (δ³⁴S) values, indicating H₂S intrusion into the plants occurred in the sampling site with the most intense root oxidizing capacity, as indicated by a greater Fe plaque formation. These apparently contradictory findings may be attributed to oxidizing capacity of root tips and root hairs sufficient to promote Fe(III) deposition in the rhizosphere, preceding deposition of plaques on the roots. While this rhizosphere oxidation may result in a more efficient sulphide detoxification during the day photosynthetic phase, root tips and hairs may serve as vulnerable sites for sulphide intrusion at night. The presence of Fe plaque on C. serrulata roots and rhizomes reveals the complexity of seagrass–sediment interactions and deserves further attention to understand if this is a local phenomenon or a newly discovered adaptive mechanism in seagrasses.     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999     

Compartmentation and Fluxes of Sugars in Roots of Phaseolus Vulgaris Under Phosphate Deficiency

The influence of phosphate deficiency on the sugar accumulation and sugar partitioning in the root cells of bean (Phaseolus vulgaris L.) was studied. Bean plants were cultured 17 - 19 d on a phosphate-sufficient and phosphate-deficient nutrient medium. Phosphate deficit in the growth medium resulted in increased sugar concentration for about 30 % in the apoplastic and cytoplasmic compartments as well as in the vacuoles of root cells. However, the distribution of sugars between apoplast and cytoplasm compartment and vacuole was not affected by decreased phosphate concentration. About 20 % of sugars were found in the apoplast and cytoplasm, about 80 % in the vacuole. Low phosphate concentration enhanced influx of exogenous ¹⁴C-sucrose into meristematic and elongation zones of root. The ¹⁴C-labelled sugar content in the root tips increased for about 60 % as compared to control plants. Phosphate deficiency increased also ¹⁴C-glucose uptake and content in the root tips. However, the amount of ¹⁴CO₂ liberated during respiration of P-deficient roots (after feeding with uniformly labelled ¹⁴C-glucose) was lower than ¹⁴CO₂ respired by control plants, thus a large part of accumulated sugars seems to be metabolically inactive. This revised version was published online in July 2006 with corrections to the Cover Date.