Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging

Background and Aims

Measuring the Al³⁺ uptake rate across the plasma membrane of intact root cells is crucial for understanding the mechanisms and time-course of Al toxicity in plants. However, a reliable method with the sufficient spatial and temporal resolution to estimate Al³⁺ uptake in intact root cells does not exist.

Methods

In the current study, fluorescent lifetime imaging (FLIM) analysis was used to quantify Al³⁺ uptake in the root-cell cytoplasm in vivo. This was performed via the estimation of the fluorescence lifetime of Al–lumogallion {5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid} complexes and measurements of intracellular pH while exposing arabidopsis seedlings to acidic and Al³⁺ stresses.

Key Results

The lifetime of Al–lumogallion complexes fluorescence is pH-dependent. The primary sites for Al³⁺ entry are the meristem and distal elongation zones, while Al³⁺ uptake via the cortex and epidermis of the mature root zone is limited. The maximum rates of Al uptake into the cytoplasm (2–3 µmol m⁻³ min⁻¹ for the meristematic root zone and 3–7 µmol m⁻³ min⁻¹ for the mature zone) were observed after a 30-min exposure to 100 µm AlCl₃ (pH 4·2). Intracellular Al concentration increased to 0·4 µm Al within the first 3 h of exposure to 100 µm AlCl₃.

Conclusions

FLIM analysis of the fluorescence of Al–lumogallion complexes can be used to reliably quantify Al uptake in the cytoplasm of intact root cells at the initial stages of Al³⁺ stress.Key words: Acid stress, Al³⁺, aluminium toxicity, Arabidopsis thaliana, low pH, fluorescent lifetime imaging (FLIM), lumogallion     

The cyclic nucleotide‐gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis

The suppression of the cyclic nucleotide‐gated channel (CNGC) AtCNGC10 alters K⁺ transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca²⁺, K⁺, Li⁺, Cs⁺ and Rb⁺ across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K⁺ in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non‐invasive ion‐specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H⁺, Ca²⁺ and Mg²⁺ in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca²⁺ influx from the external media corresponded to a lower intracellular Ca²⁺ activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H⁺ influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K⁺ and lower Ca²⁺ and Mg²⁺ content in the shoots, indicating disturbed long‐distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K⁺, Ca²⁺ and Mg²⁺ concentrations in the solution led to altered K⁺, Ca²⁺ and Mg²⁺ content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca²⁺ (and probably Mg²⁺) transport in plants, which in turn regulates K⁺ transporters' activities.     

Molecular marker linked to a chromosome region regulating seed Zn accumulation in barley

Zinc deficiency is a critical nutritional problem in soils, restricting yield and nutritional quality of barley (Hordeum vulgare L.). Some genotypes (Zn-efficient) can produce greater yield and accumulate more Zn in seed under Zn deficiency than standard (Zn-inefficient) genotypes. However, there is little information regarding the genetics of Zn uptake/accumulation and location of genes conferring Zn efficiency in barley. Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley. With the aim of developing molecular markers for increased accumulation of Zn in seed, a population of 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara 3771 (high-Zn-accumulator) was screened in the field and glasshouse for seed Zn concentration and content. One dominant DNA polymorphism was detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. The candidate MFLP marker was isolated from the MFLP gel, re-amplified by PCR, cloned, sequenced, and converted into simple sequence-specific and PCR-based marker. This marker, located on the short arm of chromosome 2H, might be useful for the improvement of barley nutritional quality and productivity programs in Zn-deficient environments. However, high seed Zn alone can not replace the need for Zn fertilization.     

Reactive Oxygen Species Production in Wheat Roots Is Not Linked with Changes in H+ Fluxes During Acidic and Aluminium Stresses

Aluminium stress induces peroxidation of lipids in the plasma membrane, the effect akin to that caused by reactive oxygen species (ROS). ROS have recently been proposed as regulators of redox-dependent ion transport across the plasma membrane during biotic and abiotic stresses, thus contributing to the plant defence system. The aim of this study was to discover whether ROS production is linked to redox-dependent H⁺ transport system located at the plasma membranes of two near-isogenic lines of wheat (Triticum aestivum L., ET8 = Al-resistant, ES8 = Al-sensitive).The activities of NADPH-dependent ROS synthase and SOD were increased in both wheat lines 15 and 30 min after Al treatments. However, the ROS production was also increased under acidic stress. There was no difference between the two wheat lines in the root-cell plasma membrane capacity to efflux H⁺ in response to potassium ferricyanide after Al and acidic treatments. In ET8, both stresses led to increases in ROS production and H⁺ influx.ROS production in wheat seedlings was activated primarily by low pH exposure rather than by the Al stress. ROS production and breakdown in wheat seedlings under Al and acidic stresses appear to be linked to the intracellular metabolic changes rather than to the increased activity of plasma membrane-based NADPH-dependent ROS synthase.Key Words: ion fluxes, reactive oxygen species (ROS), redox system, superoxide dismutase (SOD), Triticum aestivum L., wheat     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Deep placement of manganese fertiliser improves sustainability of lucerne growing on bauxite residue: A glasshouse study

Successful revegetation of bauxite residue sand (BRS) requires large inputs of nutrients such as manganese (Mn), yet Mn deficiency is still encountered, raising doubts about sustainable revegetation of BRS disposal areas. The application of deep placement of Mn, a measure common in agriculture, was examined as a method for improving productivity and sustainability when lucerne (Medicago sativa L.) is used as a species for BRS revegetation. In pots containing BRS, Mn was banded at 2.5-, 10- and 20-cm depths at rates of 10, 20 and 50 g g^–1 BRS. Two lucerne genotypes used were Salado, a Mn-deficiency-tolerant variety, and Sirosal, a Mn-deficiency-sensitive variety. Banding at 10-cm depth produced the best shoot growth of Sirosal at each Mn rate. Greatest shoot growth in Salado was found at 2.5-, 10- and 20-cm depths for 10, 20 and 50 g Mn g^–1 BRS, respectively. Deep banding 20 g Mn g^–1 BRS at 10-cm depth significantly increased lucerne growth compared with mixing through the profile. Banding at 20 cm produced Mn deficiency symptoms in lucerne during early growth, but symptoms were alleviated when sufficient amounts of roots proliferated in the banding zone. Dissolution and movement of Mn away from the fertiliser band were also investigated. In pots without plants, water throughput from watering twice weekly to 110% field capacity had no effect on the amount of extractable Mn at distances more than 1 cm away from the original Mn band position. Whilst not only providing a more effective supply of Mn for BRS revegetation over one growth period, deep-banding of adequate rates of Mn may also result in a longer residual value, reducing the need for frequent broadcast applications.     

Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus

The present study examined the effect of phosphorus (P) limitation on cluster root formation and exudation of carboxylates by N₂-fixing white lupin (Lupinus albus L. cv. Kiev) grown in a P-deficient sandy soil. Plants received 10 (limited P) or 200 g P g^–1 soil as FePO₄ (adequate P) and were grown in a phytotron at 20/12 °C (12/12 h) for 76 days in soil columns. Cluster root formation was assessed and root exudates were collected at 9-day intervals. Shoot and root dry weights were higher in plants grown in the adequate-P compared to the limited-P treatment for 67 days. No clear difference in the total root length was observed between two P treatments before day 58. However, the specific root length increased rapidly from 17 m g^–1 DW at day 40 to 28 m g^–1 at day 49 in the P-limited plants, but decreased in the P-adequate plants. The effect of P limitation on enhancement of cluster root formation was observed from day 40 and reached the maximum at day 58. The number of cluster roots was negatively correlated with the P concentration in both roots and shoots. Phosphorus limitation increased exudation of citrate from day 40. The exudation of citrate displayed a cyclic pattern throughout the experiment, and appeared related to internal P concentration in plants, particularly P concentration in shoots. The sorption of exogenously added citrate in the soil was also examined. The amount of extractable citrate remained unchanged for 2 h, but decreased thereafter, suggesting that the soil had a low capacity to sorb citrate, and the rate of its decomposition by microorganisms was slow. Collecting solution leached through a soil column is a simple and reliable method to acquire root exudates from white lupin grown in soil. The results suggest that formation of cluster roots and exudation of citrate in white lupin are regulated by P concentration in shoots.     

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.     

5, 10-Methylenetetrahydrofolate reductase (MTHFR) 677 C --> T genetic polymorphism in 228 Croatian volunteers

5, 10-Methylenetetrahydrofolate Reductase (MTHFR) is one of the key enzymes in the metabolism of homocysteine, where it catalyses its remethylation. The autosomal recessive bp 677 C --> T mutation in the MTHFR gene leads to the substitution of valine for alanine. Individuals who are homozygous for this C677T mutation exhibit a decreased specific activity and increased thermolability of this enzyme. This leads to increased plasma levels of homocysteine, which is a known risk factor for atherosclerosis and various manifestations of the atherosclerotic disease. The aim of this study was to find out the distribution and frequency of this mutation in the general Croatian population. A group of 228 volunteers (175 males and 53 females) has been analyzed for the MTHFR polymorphism, which revealed the following distribution: 105 (46.05%) individuals were without mutation (C/C), 102 (44.74%) were heterozygous (C/T) and 21 (9.21%) homozygous (T/T). These findings are within the results of studies on other European populations.