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- Materials and Methods
Micronutrient malnutrition, in particular zinc (Zn) deficiency, affects many people worldwide, especially in developing countries (Welch & Graham, 1999, 2004; Cakmak, 2008), causing various diseases (Gibson, 2006; Cakmak, 2009). Micronutrient deficiencies are generally the result of low micronutrient density in staple food (wheat, rice, maize), which represents a major proportion of the diet in developing countries (Conway & Toenniessen, 1999; Graham et al., 2007). The micronutrients in the cereal grains are not uniformly distributed. The main accumulation sites of micronutrients are the embryonic tissue and the aleurone layer, which are largely removed (polished off) during food processing, further reducing the micronutrient content of the diet. Global climate change is likely to aggravate micronutrient malnutrition in the future, as the concentration of micronutrients in cereal grains decreases with the increase in atmospheric CO2 (Manderscheid et al., 1995; Loladze, 2002; Yang et al., 2007; Högy & Fangmeier, 2008). Enrichment of staple food with micronutrients, especially Zn and Fe, through genetic or agronomic approaches, has attracted increasing attention recently (Bouis, 2003; Poletti et al., 2004; Welch et al., 2005; Nestel et al., 2006; Brinch-Pedersen et al., 2007; Cakmak, 2008, 2009; Cakmak et al., 2010).
Although substantial efforts have been made to understand Zn transport in plants (Herren & Feller, 1994; Pearson & Rengel, 1994; Pearson et al., 1995, 1996a,b, 1998, 1999; Rengel, 2001; Tauris et al., 2009; Waters et al., 2009), still no clear picture exists of how Zn is transported to ears and stored in the grains. One of the barriers to a better in-depth understanding of metal transport and accumulation in plants is the lack of appropriate methods that allow the localization of individual metals at the tissue and cellular levels (Mills et al., 2005; Grotz & Guerinot, 2006). This applies, in particular, to the quantification of the spatial distribution of mineral elements within the seeds. There are several methods available dealing with the spatial distribution of elements in seeds, but each has its limits. One approach is the mechanical separation of different seed tissue that, at best, only allows a rough estimation of major differences in tissue mineral concentrations (Bityutskii et al., 2002), even though such analysis has contributed to describing the differences among the embryo, seed coat and endosperm. A simple but powerful tool, particularly for breeding purposes, is the use of dyes for staining of specific elements, such as dithizone for Zn (Ozturk et al., 2006; Choi et al., 2007) and potassium ferrocyanide for Fe (Choi et al., 2007; Pintasen et al., 2007). However, these techniques can only provide a rough and semiquantitative estimate of metal distribution and concentration. Much more powerful combinations of X-ray analysis and electron microscopy techniques, such as energy-dispersive X-ray (EDX) analysis (Otegui et al., 2002), microproton-induced X-ray emission (micro-PIXE, Bhatia et al., 2003; Vogel-Mikušet al., 2007) and synchrotron X-ray fluorescence (S-XRF) mapping (Young et al., 2007; Meharg et al., 2008; Lombi et al., 2009), as well as high-resolution secondary ion mass spectrometry (SIMS) (Heard et al., 2001; Feeney et al., 2003; Moore et al., 2010) are currently available methods for better localization of individual components. In particular, Kim et al. (2006) convincingly demonstrated the potential of combining S-XRF microtomography in relating molecular studies on micronutrient transport to mapping element distribution in Arabidopsis seeds. By using a more precise laser capture microdissection technique and gene expression profiling, Tauris et al. (2009) proposed a roadmap for zinc trafficking in the developing barley grain.
Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is an emerging technique for measuring metal distribution in plant tissues (Becker et al., 2008; Shi et al., 2009; Wirth et al., 2009; Wu et al., 2009). The advantages over other methods might be the ease of sample preparation and handling, the low cost and, most importantly, the possibility of tracing the movement of the mineral elements using stable isotopes introduced to the plant at specific developmental stages with high sensitivity.
Ear culture is an established method for growing detached ears of small grain cereals in culture medium (Donovan & Lee, 1977; Herren & Feller, 1994; Pearson et al., 1996a,b; Zhou et al., 2006). This method is particularly suited to metals at specific developmental stages during reproductive growth. In this experiment, we grew detached wheat ears in the culture medium with the 70Zn isotope, in order to trace the movement of the applied Zn to the grain. Using LA-ICP-MS, the spatial distribution of Zn within the grains was studied in order to elucidate the Zn transport pathway within the developing grain.
Materials and Methods
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- Materials and Methods
The seeds of the spring wheat Triticum aestivum L. cv ‘Segetoria’, kindly supplied by I. Cakmak, Sabanci University, Turkey, were sown in Micherlich pots, each pot containing 4 l peat substrate (Einheitserde type P, 10% clay, bulk density 500 g l−1). The plants were grown in a glasshouse with natural light and a temperature regime (day : night) of 25 : 20°C (aeration : heating set). The plants were watered and fertilized regularly and treated with fungicides or insecticides when necessary. At the beginning of anthesis, individual ears were tagged to be able to select and compare ears at exactly the same developmental stage.
Wheat ear culture
Detached wheat ears were cultured according to Singh & Jenner (1983) and Sharma et al. (1995). At anthesis (Expt 1) or 3 wk after anthesis (Expt 2), the tagged ears with the flag leaf removed were cut at the penultimate stem for in vitro culture. The stems were sterilized in 70% ethanol for 1 min and 1% NaClO for 5 min, and then washed with sterile distilled water. After sterilization, the ears were further cut c. 2 cm from the bottom of the penultimate stem under sterile distilled H2O and then transferred into a cotton-plugged conical flask containing 100 ml liquid culture medium. The basic components of the culture medium were as in the MS culture medium (Murashige & Skoog, 1962) with modifications in nitrogen and glutamine concentrations according to preliminary experiments aimed at matching grain growth rates of intact ears in the pots (Table 1). Zn concentration and form in the culture medium were varied according to the treatments.
Table 1. Composition of the wheat ear culture medium
|Class of nutrient||Ingredient||Concentration (mg l−1)|
|ZnSO4.7H2O||8.6, 17.2, 34.4, 68.8 or 25.8|
| ||Nicotinic acid||0.5|
| ||Sucrose||50 000|
Expt 1 was designed to investigate how the Zn concentration in the culture medium affects Zn uptake and partition in detached wheat ears at the early growth stage of grain setting. Treatments represented normal to high Zn concentrations of 30, 60, 120 and 240 μM in the form of natural isotopes of ZnSO4. The ear culture started at anthesis and the treatment duration was 7 d.
In Expt 2, we studied how late-supplied Zn was transported to wheat ears and distributed in the grains by using the stable isotope 70Zn. Natural isotopes of Zn and the 99% enriched stable isotope 70Zn (STB Isotope Germany GmbH, Hamburg, Germany) in the form of ZnSO4 were used in the study. Zinc concentrations of 30 and 90 μM were used to represent normal and high Zn supply. Ear culture was started at 21 d after anthesis (DAA) and the treatment duration was 9 d.
All media and containers were autoclaved. Detached ears were cultured in conical flasks and incubated in a growth cabinet under a day : night temperature cycle of 25 : 22°C, a 16 h photoperiod and a photosynthetically active radiation (PAR) of 120 μmol m−2 s−1.
Zn analysis of different organs of the wheat ear
After termination of the ear culture, the wheat ears were separated into grains, rachis, glumes (including the lemma and palea), peduncle and the node right below the peduncle. From each ear, three uniform grains in the middle position were selected for LA-ICP-MS analysis. The other grains and organs from the same ear were oven-dried at 60°C for 3 d and the dry weights were recorded. The dried samples were ground by a vibrating cup mill (Puverisette; Fritsch GmbH, Idar-Oberstein, Germany) and used for total Zn analysis.
The samples were dry-ashed in a muffle oven at 480°C for 16 h. The ash was dissolved in 1 ml of 1 : 3 (HNO3 : H2O) ultrapure nitric acid, diluted with further 9 ml of deionized water. After passing the solution through filter paper (Rotilabo-Rundfilter AP52.1, 180s; Carl Roth GmbH, Karlsruhe, Germany), the solution was analysed by ICP-MS (7500cx; Agilent Technologies, Santa Clara, California, USA) for the isotopes 64Zn, 66Zn, 67Zn, 68Zn and 70Zn, separately.
Localization of Zn in grains using LA-ICP-MS
Sample and calibration standard preparation Fresh grains were freehand-sectioned. The cross-sections were fixed on glass slides using double-face fotostrip (Tesa AG, Hamburg, Germany) and subsequently air-dried for at least 24 h before analysis.
For calibration, wheat flour (10 g) was spiked with different Zn standard solutions (10 ml) and dried at 60°C for 48 h. The Zn-spiked flour was homogenized using a vibrating cup mill (Puverisette; Fritsch GmbH) for 10 min. The flour was then pressed into pellets of 5 mm diameter. The Zn concentration was determined by digesting the flour in nitric acid and measuring by ICP-MS (7500cx; Agilent Technologies).
For calculation of the grain Zn concentration, the Zn : 13C ratio (13C as internal standard) of the calibration standards and the grains were used. Four cross-sections from four grains of each treatment were analysed; the images shown in Fig. 5 and Fig. 6 are representative of four independent measurements of each treatment.
Zn localization A quadrupole ICP-MS coupled to a laser ablation system, UP193SS (New Wave Research, Fremont, CA, USA), was used to determine the Zn concentration and distribution in wheat grains. Samples and matrix-matched calibration standards were arranged in a laser ablation chamber (Supercell; New Wave Research) and ablated under the same experimental conditions, in order to allow the calculation of Zn concentrations. The ablated material was transported by argon as a carrier gas to the plasma. The ICP-MS instrument tuning was optimized with respect to the maximum ion intensity of low masses. The flow rate of the carrier gas and the make-up gas were adjusted to optimum output; the flow rate was 0.25 l min−1 for the carrier gas and 1.25 l min−1 for the make-up gas. Radiofrequency (RF) power was set to 1300 W and the reaction mode was off. The laser parameters were set to 2 J cm−2 of output energy, 10 Hz repetition rate, 50 μm diameter of the crater size, and 20 μm s−1 scan speed. Measured elements were 64Zn, 66Zn, 67Zn, 68Zn, 70Zn and 13C. 13C was used as an internal standard to normalize for different quantities of seed tissue ablated.
For the calculation of 70Zn concentration in the endosperm of seed cross-sections, two locations (two lines in the length of 500 μm) from both sides of the crease of each cross-section were chosen. The 70Zn concentration of the endosperm was the mean of 200 measurements obtained by LA-ICP-MS run at the speed of one measurement per 5 μm. Three cross-sections per treatment were measured.