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In the past hundred years, plant monitoring has attracted widespread attention from environmental and ecologists because of its low cost, intuitive effect and high sensitivity. Air pineapples are a special class of plants that grow in the air and do not require soil, and the water and nutrients needed for their growth can all come from the air, sometimes referred to as "air plants". They have real roots, but the roots are underdeveloped, have no function of absorbing water and nutrients, only play the role of fixing the plant and a small amount of air exchange, and the scales on the surface of the leaves are considered organs where they really absorb water and nutrients.
Because of this, they can also absorb pollutants (including organic pollutants and heavy metal pollutants) in atmospheric dust precipitation at the same time, thus becoming effective "indicator plants" for monitoring environmental changes. As early as 1952, MacInland used Tillandsia usneoides to detect fluoride levels in rainwater. Calasansa et al. also found that Pinluo is a good indicator plant for detecting mercury pollution of heavy metal ions in the air, because it can quickly and effectively accumulate mercury ions floating in the air, and has strong antagonism to some stressful environments, such as high temperature, high mercury ion concentration and oxidant (Cl2). Eduardo et al. used two other species of air bromeliads, T. capillaris and T. permutata, as indicator plants, and found that they could also effectively detect the presence of heavy multi-elements such as V, Mn, Fe, Ni, Cu, Zn, Pb and Br.
However, so far, most of the studies on the use of air bromeliads to monitor heavy metal pollution have focused on the effect of plant enrichment of heavy metals, while few studies have explored the specific mechanism of heavy metal enrichment in air bromeliads. Only Filho et al. used Pingluo Hollows as an indicator plant for heavy metal pollution, and used scanning electron microscopy and X-ray fluorescence technology to analyze the enrichment site and content of mercury, and found that the scales on the stem and leaf surface of Songluo were highly adsorbed of mercury, and rarely adsorbed on epidermal cells, while mercury was not found in the leaves of Songluo.
However, the scales on the surface of air bromeliads are composed of outer wing cells, intermediate ring cells and internal dish cells, and which type of cells absorb heavy metal elements more effectively has not been studied. Therefore, we selected Tillandsia stricta 'Hard leaf', which is more common in air bromelias, as the material, and after stressing it with lead, the most typical pollutant in the environment, we explored the enrichment effect of air bromeliads on lead by atomic absorption and other methods, and explored the specific mechanism of lead enrichment in air bromeliads by scanning electron microscopy and energy spectroscopy.
Materials and methods
Fifteen sclerophyllum sclerophyllum with a consistent plant type were selected from the cultivation greenhouse as research materials. The cultivation conditions were as follows: day/night: 14 h/10 h, temperature 25°C, humidity 70%~80%, light intensity 14 400 lux.
Lead stress was performed on Sclerophyllum sclerophyllum plants in the form of PbNO3 solution, and five gradients were set up, and the lead treatment concentrations were 0, 1, 10, 50, and 100 mg/L, respectively, and each concentration was treated with 3 replicates. Referring to the normal watering method of Sclerophylla Hollow, Sclerophyllum Hollows was completely submerged in different concentrations of lead solution at the same time every day for 2 min each. After 15 days, the stressed pineapple leaves were taken for the determination of heavy metal content.
The samples were digested by wet digestion: 2.0 g of pineapple leaves were accurately weighed, dust and other adhesions were rinsed with distilled water, placed in a constant temperature drying oven, killed at 105°C for 30 min, and dried to constant weight at 50°C. Cut the leaves as much as possible with scissors, take 0.2 g into a long-necked round bottom flask, add 21 mL concentrated nitric acid and 7 mL perchloric acid, and heat it with an electric jacket for wet digestion. Pour the digested solution into a 25 mL volumetric flask and set the volume with distilled water.
Samples are determined with flame atomic absorption spectroscopy (AAS): the sample is appropriately diluted according to the treatment concentration. The analysis conditions of Pb were as follows: wavelength 283.3 nm, lamp current 10 m A; The spectral pass band was 0.7 nm, and the auxiliary gas was air, 17 L/min; Gas is acetylene, 1.4L/min; The measurement method is AAS; The background deduction method is the Saman method.
Scanning electron microscopy (SEM) sample preparation and observation
The leaves of the same part of the sclerophyllum were taken and fixed with 4% glutaraldehyde for 48 h after cleaning, washed with distilled water, and dehydrated with 30%, 50%, 70%, 80%, 90%, 100% (2 times) gradient concentration alcohol for 10min each. Immediately after dehydration, the sample is fixed on the rupture plate and left to dry naturally in a closed container. Ion sputterer coating, Leica S440 scanning microscope observation, photographed.
Energy Spectroscopy (EDS)
In the SEM field of view, select a representative intact scale. At different locations of the pterygoids, ring cells and dish cells of the scale, a fixed point is set for spot scanning to analyze the main elements and lead content of the spot. One-way ANOVA was performed with SPSS 18.0 statistical software to compare the significance of differences between treatments.
Sclerophyllum sclerophyllum was stressed with different concentrations of PbNO3 solution every day for 15 days, and there were no obvious death symptoms in all plants after the end of the experiment. The plants treated with 0~10 mg/L PbNO3 solution remained green, but the plants treated with 50 mg/L and 100 mg/L PbNO3 began to have yellowing of leaves, and the leaf tips began to dry up, and the leaves of 100 mg/L PbNO3 treated plants turned yellow and dried up.
The presence of lead was also detected in Sclerophyllum sclerophyllum plants without lead stress, which could reach 0.31μg/g. With the increase of pollutant concentration, the more air bromeliad leaves adsorb and accumulate pollutants (Table 1). Statistical analysis showed that the adsorbed and accumulated lead content of Sclerophyllum sclerophyllum increased significantly with the increase of treatment concentration under the stress of PbNO3 solution (p<0.05).
Table 1
Adsorption characteristics of lead by sclerophyllum hollowleaf surface scales
Scanning electron microscopy (SEM) showed that the sclerophylla hollow leaf table was covered with white scales and had a special "sunflower" shape. The scales are composed of three types of cells: pterygocytes, ring cells, and dish cells (Figure 1). The scales are composed of 3 layers of cells, and the central layer has 4 cells, which are dish-shaped, that is, disc cells; Outside the dish cell is surrounded by a layer of cells, a total of 8, namely ring cells; The outermost layer of the scales are long wing cells, which are tightly connected to each other and have serrated edges. Energy profiling (EDS) showed that C, N, O, MG, Al, Si and Ca, which are essential for plants, were detected on all three types of cells, with C and O accounting for the highest proportion.
The presence of lead was detectable in leaf surface scale cells of Sclerophylla Hollow plants without lead stress treatment (Fig. 1) and in plants treated with lead nitrate solution (Fig. 2). Regardless of the concentration of Sclerophyllum sclerophyllum plants, the proportion of lead was always the lowest among the outermost pterygocytes, the second lowest in the middle ring cells, and the highest in the innermost dish cells (Table 2).
Table 2
As an indicator plant for pollution monitoring, it usually needs to have the following two basic conditions, one is sensitive to pollutant response, even at very low pollutant concentrations, it can show obvious characteristics in form or physiology, so as to reflect the concentration change of pollutants. Second, it has a strong resistance to pollutants and will not be killed soon after being polluted. Experiments showed that the lead content enriched by Sclerophyllum sclerophyllum increased significantly with the increase of lead solution concentration (Table 1), indicating that this air bromeliad could effectively adsorb and enrich lead. Moreover, the presence of these pollutants can be quickly detected in its leaves under the treatment of very low concentrations of lead, which shows its ability to quickly and effectively monitor pollutants, thus having the first basic conditions as an indicator plant.
In addition, under different concentrations of lead treatment, sclerophylla air bromeliads show strong anti-pollution ability, even at very high pollutant concentrations (100 mg/L), this air pineapple can survive for more than half a month. In nature, the concentration of lead in polluted atmosphere and water is rarely 100 mg/L. Therefore, it can be said that sclerophyllum air pineapple has strong resistance to lead pollution, and has the second basic condition for monitoring the pollution of heavy metal elements such as lead. Although we treat air pineapples with a lead solution, consider that lead is always present as ions when absorbed by plants, both in air and in solution. Therefore, it is also feasible to apply air pineapples with strong lead absorption capacity in solution to air lead pollution monitoring.
The effective enrichment of pollutants such as lead is closely related to its unique biological characteristics. As a special plant that can survive in the air, air pineapples must rely on their leaves to absorb moisture and nutrients in the air, and their leaves have a strong absorption capacity, which is also proved by many studies. The structure of air bromeliads that absorb water and nutrients are scales on the surface of the leaves.
The study of the absorption of Hg particles by microscopic technology on the absorption of Hg particles by pineapple air bromeliad also showed that most of the Hg particles were adsorbed by leaf surface scales, and a small part were adsorbed by epidermal cells, while no Hg was found in the mesophyll cells inside the leaf. Our study also shows that the presence of lead can be detected in the leaf surface scales of Sclerophyllum sclerophyllum regardless of the concentration of lead solution. It further proves that leaf surface scales are not only the main structure of water and nutrient absorption, but also the main enrichment structure of heavy metal elements.
Energy spectrum analysis showed that the three types of cells of air bromeliad leaf episcale played different roles in enrichment of lead, and the content of lead in the innermost dish cells was greater than that of the middle ring cells and larger than that of the outermost pterygocytes (Table 2). The airborne bromeliad leaf surface scales are sunflower-shaped and connected to the mesophyll cells inside the leaf through several channel cells. In mature scales, the 3 types of cells in the scales first absorb water, nutrients and other substances in the external environment, and then transport them to mesophyll cells through channel cells, which is not a passive absorption process, but an active absorption process, otherwise the absorbed water and nutrients and other substances will flow back to the external environment.
In addition, histochemical studies on maize (Zea mays) showed that the uptake of lead by young maize roots was similar to that of plants for beneficial mineral elements, and the increase of ATPase and acid phosphatase activities in parenchymal cells in plants indicated that lead migration required energy consumption and was a metabolic active absorption process. Therefore, although we soak it in lead solution when we stress Sclerophyllum hollow, and the three types of cells in the scales have equal opportunities to contact lead particles, active absorption makes lead have a tendency to migrate from the outermost pterygoid cells, the middle ring cells to the innermost dish cells, so that the innermost cells contain the largest amount of lead.