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Chinese Science Bulletin 2005 Vol. 50 No. 24 2843—2849 As concentration in the pinna of the plant may exceed 10000 mg/kg, which is much higher than the concentra- Subcellular distribution tion of phosphorous, an essential macronutrient element in plants[4,5]. Investigation of the mechanisms underlying the and compartmentalization elevated As uptake, transport and distribution in P. vittata L. may help us to realize the mechanisms of As enrich- of arsenic in Pteris vittata L. ― ment and tolerance in plants[6 12], which is critical to understand the evolution of this unique capacity of As hy- CHEN Tongbin, YAN Xiulan, LIAO Xiaoyong, peraccumulation and to improve its potential in field ap- XIAO Xiyuan, HUANG Zechun, XIE Hua plication. & ZHAI Limei Investigating elemental microdistribution in hyperac- Center for Environmental Remediation, Institute of Geographic Sciences cumulating plants also helps illuminate the physiological and Natural Resources Research, Chinese Academy of Sciences, Beijing function of that element in plants, which may ultimately 100101, China Correspondence should be addressed to Chen Tongbin (email: chentb@ help identify the mechanisms of accumulation and detoxi- igsnrr.ac.cn) fication of heavy metals. Previous studies in our labora- tory using transmission electron microscopy (TEM) and Abstract The subcellular distribution of arsenic (As) in environmental scanning electron microscopy (ESEM), Pteris vittata L., an As-hyperaccumulator, was studied to de- were unable to fully describe As microdistribution in P. termine As compartmentalization and to explore the mechanisms that confer As tolerance. When the plant was grown in vittata L. because the average concentration of the plant a nutrient solution without additional As, most of the accu- (up to 4000 mg/kg) was lower than the lowest detection mulated As was isolated to the cell wall. However, in plants limits of the equipment. Although energy depressive X-ray growing in a nutrient solution containing 0.1 or 0.2 mmol/L analysis (EDXA) and synchrotron radiation X-ray fluo- As, approximately 78% of the total As accumulated within rescence (SRXRF) may provide lower As detection limits, the pinna. The proportions of As accumulation in the cyto- these techniques cannot localize and quantify As at a sub- plasmic supernatant fraction were 78% of that in the pinna cellular level because the light field of the X-ray scan is and 61% of that in the plant. In either treatment group (0.1 too large. Chen et al. studied As distribution in the pinna or 0.2 mmol/L As), the fraction containing the lowest level of of P. vittata L. using SRXRF technology, and reported that As was the organelle fraction. These results suggest that As accumulates in the pinna where it is primarily distributed in the concentration of As within the midrib was higher than the cytoplasmic supernatant fraction. The role of As com- on the two pinnate sides, and the greatest difference in partmentalization may be intricately linked with As detoxi- distribution was found in the pinnate tip, indicating that fication in P. vittata L. the plant had the ability to transport As from the xylem into the mesophyll[6]. Using EDXA analysis, Lombi et Keywords: arsenic (As), compartmentalization, detoxification, hy- [11] peraccumulator, Pteris vittata L., subcellular distribution. al. reported that As was primarily distributed within the upper and lower epidermis of the pinna in P. vittata L. DOI: 10.1360/982005-943 However, using SRXRF, Huang[7] found the highest concentration of As in the P. vittata L. pinna was within the Arsenic (As) is a poisonous metalloid element that in- paraxial mesophyll tissue (palisade tissue) and decreased hibits plant growth and development, and may even cause in concentration from the center to paraxial regions. A plant death. In general, common plants are able to avoid similar As distribution has also been found in another As- taking up As from the surrounding medium and restrict As hyperaccumulating plant, P. cretica, indicating that most [1] transport to their aboveground biomass . Normally, As of the absorbed As was enriched within mesophyll cells, concentrations in the aboveground biomass of common and the As concentration in epidermal cells was relatively plants range from 0.01 to 5 mg/kg DW and the As trans- low[13]. These studies have described the microdistribution port coefficient (As concentration of the aboveground of As within As-hyperaccumulators; however the subcel- biomass vs. that of the underground biomass) is usually lular distribution of As in P. vittata L. remains unknown. less than 1[2]. However, As-hyperaccumulating plants A quantitative assessment of As concentrations in the demonstrate an excellent ability to tolerate the toxicity of subcellular plant fractions isolated by centrifugal separa- As and to transport As to their aboveground regions. In- tion can allow for a more detailed study of As distribution deed, high As transport coefficients are observed whether in hyperaccumulating plants. Previous studies have suc- As-hyperaccumulators are grown in the greenhouse or in cessively used this method to evaluate the subcellular dis- the field. For Pteris vittata L., the first identified As- tribution of heavy metals in plants[14―18]. For example, hyperaccumulating plant, the As transport coefficient has using subcellular fraction separation, Wan et al.[15] re- been found to be quite high (ranging from 1 to 7)[3]. The ported differing shoot/root Cd partitioning in two wheat As concentration in P. vittata L. may be hundreds of genotypes. Previous work by Ramos et al.[18] used similar thousand times higher than in common plants[3], and the methods to evaluate the subcellular distribution of Cd as

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ARTICLES well as Cd-Mn interactions in Lactuca sp. In order to mmol/L cysteine. The final pH of the solution was ad- identify the subcellular distribution of As and its relation justed to 7.8. All homogenizations and subsequent frac- to As tolerance and detoxification, the present study util- tionations were performed at 4℃. The resulting debris ized centrifugal separation to evaluate As concentrations was washed three times with the grinding medium. The in subcellular fractions of P. vittata L. homogenate was brought up to a volume of 15―20 mL 1 Materials and methods and transported into a 50 mL centrifugal tube, then centrifuged at 300 g for 30 s using a high speed refrigerated 1.1 Experimental design centrifuge. The pellet was considered the cell wall fraction. The nutrient solution, modified according to the The supernatant was further centrifuged at 20000 g for 45 Hoagland nutrient solution protocol, contained (mmol/L): min to separate the cell organelles; thus the pellet from N 4.8, P 0.4, K 2.0, Ca 2.5, Mg 0.25, Na 0.2, Fe 2.24×10−2 , this second centrifugation was considered the cell organ- Mn 2.3×10−3 , B 1.15×10−2 , Zn 1.9×10−4 , Cu 8×10−5, Mo elle fraction. The resultant supernatant solution was con- 5×10−5, Cl 0.2, and S 0.28. There were three experimental sidered the cytoplasmic supernatant fraction including groups: treatment with 0 (control), 0.1 or 0.2 mmol/L As. macromolecular organic matter and inorganic ions in the [16] All experiments were replicated 4 times. The pH of the cytoplasm and vacuoles . Under a microscope, the cell nutrient solution was adjusted to 6.5 using 0.1 mol/L of wall fraction contains fibrous structures (Fig. 1(a)); the NaOH or HCl. organelle fraction contains clumpy green material and many elliptical green structures dissociated in buffer solu- 1.2 Plant culture tion (Fig. 1(b)). The cytoplasmic supernatant fraction ap- P. vittata L. spores were collected from the Hunan pears as clear and transparent colloidal matter. Province in China and sprinkled onto plastic trays covered 1.4 Chemical analysis with a plastic cling film to maintain suitable moisture. The The dried samples were ground to a fine powder. 10 plants were then cultured in a greenhouse. Four plant mL of HNO and 0.5 mL of HClO was added to 0.1―0.2 seedlings at the 5―6 frond stage were transferred to plas- 3 4 g of the powdered samples and incubated overnight. The tic pots. The pots were loaded with siliceous sand which samples were then digested until the solution became clear, had been treated with HCl for 24 h, washed with tap water and the solution was then brought up to 50 ml with dis- and rinsed twice with deionized water. The diameter of [21] tilled water . The cell wall, organelle and cytoplasmic each pot was 12 cm and the height was 10 cm. The plants supernatant fractions were digested using the methods were cultured in an environment-controlled growth described above. The As concentration within the final chamber (RXZ-300C) under the following conditions: 14 solution was determined using an atomic fluorescence h:10 h (light:dark), a day/night temperature of 26/20℃, spectrometer (Haiguang AFS-2202, China). Standard ref- and 85% relative humidity. The plants were grown for 30 erence plant (GBW-07603) was added for the QA/QC d, after which the As solution was added into the nutrient program. The amount of As recovered in the process of solution. The nutrient solution was replaced every week in subcellular fraction separation ranged from 91% to 105%. order to maintain the desired As level, and 50 mL Lof fresh nutrient solution was added every three days. In or- 1.5 Statistical analyses der to compensate for any weight loss, 80 mL of water Statistical analysis was performed using the PROC was added every other day. The plants were harvested at ANOVA and PROC GLM procedures available in SAS the end of the experiment (3 months post-transplantation). 6.0. Results were considered significant when p< 0.05. 1.3 Separation of the subcellular fractions of the pinna, 2 Results petiole and root 2.1 Subcellular distribution of As in P. vittata L. After being washed thoroughly with tap water followed Arsenic concentrations in the subcellular fractions of by deionized water, the plants were separated into pinna, the pinna, petiole and root are presented in Table 1. In the petiole and root. A fresh sub-sample of 0.5000 g of each control group, As concentrations in the three fractions of region was prepared to separate the subcellular fractions. the root were very low, approximately 1 mg/kg. However, The residual sample was dried at 60℃ for 24 h to deter- As accumulation in every fraction of the root of the plants mine the total As concentration. Subcellular distributions supplied with 0.1 mmol/L of As was significantly en- of As were determined according to the methods described hanced. Notably, As concentrations in the cytoplasmic by Hans[19] and Pathore et al.[20]. Samples of root, petiole supernatant fraction was 425 times the levels in the con- and pinna were homogenized with pre-cooled homogeni- trol-treated plants; the smallest increase in As concentra- zation solution using a glass grinder. The homogenization tion was found in the cell organelle fraction, which con- solution contained 0.25 mmol/L sucrose, 50 mmol/L tained approximately 71 times that of control levels. As Tris-maleate buffer (pH=7.8), 1 mmol/L MgCl2, and 10 arsenic concentration in the medium was increased from

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Table 1 Arsenic concentrations in the subcellular fractions of P. vittata L.a) −1 As added/ As concentration/mg·kg DW −1 mmol·L cytoplasmic supernatant cell wall cell organelle total Root 0 1.3±1.3 Ba 1.4±1.0 Ca 1.0±0.2 Ca 3.4±1.7 C 0.1 554.1±79.4 Aa 297.6±68.4 Bb 70.9±8.2 Bc 895.9±95.1 B 0.2 811.0±381.6 Aa 564.1±129.4 Aa 326.3±72.7 Ab 1770±406 A Petiole 0 2.8±0.8 Ba 3.3±2.7 Ba 1.1±0.7 Bb 8.6±2.1 B 0.1 963.8±79.6 Aa 305.9±35.0 Ab 100.2±19.4 Ac 1301±112 A 0.2 1014 ±16 Aa 442.2±134.4 Ab 98.4±24.1 Ac 1617±155 A Pinna 0 4.8±0.1 Ca 7.9±3.3 Ca 2.3±1.6 Cb 14.7±3.2 C 0.1 3019±306 Ba 683.5±274.5 Bb 158.8±57.6 Bc 4016±549 B 0.2 4813±12 Aa 1035±64 Ab 274.3±83.3 Ac 5634±71 A a) Data are shown as mean confidence interval (p< 0.05) of four replicates. Different capital letters in the same line indicate significant differences between As treatments (p< 0.05). Different small letters in the same row indicate significant differences between each cell fractions (p< 0.05).

Fig. 1. Photomicrographs of the cell wall (a) and organelle (b) fractions obtained by subcellular fraction separation (200×).

0.1 to 0.2 mmol/L, As concentrations in the cell wall and trations in the same two fractions of the petiole were cell organelle fractions further increased, but no signifi- lower than those in the root of the same treatment and cant enhancement was found in the cytoplasmic super- were similar to those in the petiole with 0.1 mmol/L of As. natant fraction. One possible explanation for this may be After treatment with 0.2 mmol/L As, the average petiole the storage limitation of As in the cytoplasmic supernatant As concentration of the cytoplasmic supernatant fraction fraction of the roots――As enrichment could not be in- was 2.6 and 10 times As concentration than that of the cell creased if its accumulation exceeded the fraction’s storage wall and cell organelle fractions, respectively. capacity. When 0.1 or 0.2 mmol/L of As was added to the me- Petiole, a “channel” through which As can be trans- dium, As concentrations in the cytoplasmic supernatant ported from root to pinna, is also capable of storing As. In fraction of the pinna were 3019 and 4812 mg/kg, respec- the 0.1 and 0.2 mmol/L treatment groups, the average As tively. Notably, after 0.1 or 0.2 mmol/L treatment, the As concentration in the cytoplasmic supernatant fraction of concentration in the cytoplasmic supernatant fraction of the petiole was 1.5 times greater than in the root. How- the pinna was 5.4 and 5.9 times greater than those of root, ever, there were no significant differences in As concen- and 3.1 and 4.7 times greater than those of petiole, respec- tration in the cytoplasmic supernatant fraction of the peti- tively. In the pinna of P. v itta ta L., As concentrations in ole between the 0.1 and 0.2 mmol/L As treatment groups. the cell wall fraction increased to approximately twice the In the 0.1 mmol/L of As treatment, the As concentrations concentration of the root, but As concentrations in the cell in the cell wall and cell organelle fraction of the petiole organelle fraction remained relatively low. were slightly higher than concentrations in the root. In the two As treatment groups, the As concentration in However, when 0.2 mmol/L of As was added, As concen- each fraction decreased in the following pattern: cyto-

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Fig. 2. Percentage of As accumulation in the various subcellular fractions of P. vittata L. treated with 0.1 and 0.2 mmol/L of As. plasmic supernatant >> cell wall >> cell organelle. Im- root and petiole was very low, only about 2.1% and 0.7%, portantly, in the pinna, the storage capability of As in the respectively, of the total As in the entire plant. cytoplasmic supernatant and cell wall fractions was much Fig. 3 shows the relative rate of As accumulation (RRA; greater than in the corresponding fractions of the root or equals accumulation of As in various subcellular frac- petiole. tion/total As accumulation in pinna, petiole or root) in the 2.2 Corresponding proportion of As distribution in sub- three treatment groups. In the control treatment, As distri- cellular fractions bution did not differ much between each fraction, and the RRA was all approximately 35%. In the pinna as well as Fig. 2 represents the percentage of As accumulation in in the petiole, the RRA of the cell wall was the highest the various subcellular fractions of P. vittata L. treated (46% and 53%, respectively) among the three fractions with 0.1 and 0.2 mmol/L As. The rate of As accumulation and for both regions the RRA of the cell organelles was in the various fractions decreased from the upper to the near 15%. In the plants treated with As, there was a much lower plant regions. The pinna were able to accumulate greater proportion of As localized within the cytoplasmic 78% of the total As throughout the entire plant, which was supernatant fraction and the RRA was 54%, 68% and 78% 7 times greater than the levels of As accumulated within in the root, petiole and pinna, respectively, with the the petiole or root. Approximately 61% of the total As in amount of As increasing gradually from the root to the the entire plant was localized within the cytoplasmic su- pinna. In contrast, the RRA of the cell wall and cell or- pernatant fraction of the pinna. Therefore, the cytoplasmic ganelle fractions decreased gradually from the root to supernatant fraction of the pinna comprises the primary pinna. In the root, the RRA of these two fractions were storage site of As. Arsenic distribution in the cell wall of the pinna accounted for 13% of the total As levels in the 32.8% and 13.5%, while they were 17.3% and 4.3% in the entire plant, which was close to the sum of As accumula- pinna, respectively. These results indicate that the cell wall tion within the three fractions of the petiole or root. The exhibited a preferential affinity for As in P. vittata L. with cytoplasmic supernatant fractions in the petiole and root low As levels. However, in plants that have accumulated a accumulated 7.7% and 5.2% of the total As, respectively. large amount of As, As is primarily distributed throughout Arsenic accumulation in the cell organelle fraction of the the cytoplasmic supernatant fraction. Interestingly, the As

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subcellular distribution of As in P. vittata L. and the results presented here indicate that As primarily accumulates in the cytoplasmic supernatant fraction of the pinna. Specifically, 78% of As within the pinna was found in the cytoplasmic supernatant fraction, and, moreover, 61% of the total As in the entire plant accumulated in the cytoplasmic supernatant fraction of the pinna (Figs. 2 and 3). Therefore, this cytoplasmic fraction represents a key site for the accumulation and detoxification of As in P. vittata L. A more detailed study of various As species and transport mechanisms in the cytoplasmic supernatant fraction should be carried out to better understand how hyperaccumulating plants endure such high levels of As. A greater understanding of the subcellular distribution of heavy metals should help identify the effects of these elements on cellular activities as well as help define their possible biochemical roles. The results presented in this paper indicate that the cytoplasmic supernatant fraction of the pinna is the main site of As distribution in P. vittata L. The cytoplasmic supernatant fraction is composed mainly of cell and vacuole sap. Cell sap is the main site for cell metabolism[25], and vacuoles primarily participate in water metabolism and provide a place to store the cellular waste as well as the by-products of cell metabolism[26]. Hall[27] suggested that the concentrations of heavy metals in cell sap should be lower than that in vacuole sap to assure normal cell metabolism. Previous studies have shown that vacuole compartmentalization may play an important role in the detoxification of heavy metals in hyperaccumulators[28―30]. In Alyssum serpyllifolium, a Ni-hyperaccumulator, 72% of the Ni accumulated by the plant was stored Fig. 3. Relative amount of arsenic in the subcellular fractions of P. in its vacuole[28]. A comparative study on the vacuole vittata L. compartmentalization of Ni reported that the hyperaccu- concentration in the cell organelle fraction appears to be mulator Thlaspi goesingense Hálácsy accumulated ap- consistently maintained at a relatively low level. proximately 2 times more Ni in the vacuole of its leaves than did the non-hyperaccumulator T. arvense L. under Ni 3 Discussion exposure conditions[29]. T. caerulescens, a Zn-hyperac- In general, As transport in P. vittata L. tends to occur cumulator, mainly accumulated Zn within the vacuoles of from underground regions to the aboveground regions, epidermal leaf cells[30]. As differs from the cations (Ni and and greater than 90% of As taken up from media is simi- Zn) in that it is a metalloid element and usually presents as [12,22] [23] larly transported upward . Liao and colleagues re- an inorganic anion in As-hyperaccumulating plants[7,11]. ported that As accumulation in P. vittata L. increased We speculate that the As in the cytoplasmic supernatant gradually from underground structures to aboveground fraction was mainly isolated by the vacuoles. Therefore, structures such that the concentration of As in the root < vacuole compartmentalization may buffer the effects of petiole< pinna; similar findings were obtained in the pre- high levels of As on cell metabolism to ensure normal sent study. A recent study with potted P. vittata L. re- growth and development of the hyperaccumulating plant. ported that 75%―80% of the total As taken up from the In P. vittata L., the cell wall appeared to play an impor- surrounding medium was stored in the pinna[24]. Similarly, tant role in retaining As. When the plant contained low Tu et al.[22] found high concentrations of As in the meso- levels of As, the cell wall could effectively isolate As and phyll tissue fraction of the pinna, whereas the As concen- prevent it from entering the cell. Even when a large tration in epidermal cells was relatively low. In addition, amount of As was enriched in the plant, more than 30% of there exists a unique organel-tricome in the frond of P. the As in the root was retained by cell wall, and in the vittata L., and the relative weight of As in the pinnate pinna, approximately 17% of the As was distributed in this trichome was 2―4 times greater than in the epidermal and fraction. Ernst et al.[31] concluded that whether the cell mesophyllous cells[8]. The present study investigated the wall could effectively constrain and detoxify heavy metals

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ARTICLES had been a controversial one. However, increasing evi- National Natural Science Foundation of China (Grant No. 40232022), dence has demonstrated that non-protoplast structures, and the National High-Tech R & D Program (No. 2003AA645010). which are mainly comprised of the cell wall, can protect References plants against the stress of heavy metals. This is because 1. Berry, W. L., Plant and factors influencing the use of plant analysis amylase and protein molecules in the cell wall contain as a tool for biogeochemical prospecting, in Mineral Exploration: large amounts of metal ion coordination groups, such as Biogeological Systems and Organic Matter (eds. Carlise D, Berry hydroxide, carboxyl, aldehyde, amidogen and phos- W L, Kaplan I R, et al.), New Jersey: Prentice-Hall, Englewood phate[32], which may interact with metal ions to isolate the Cliffs, 1986, 5: 13. metals within cell wall, reduce their cross-membrane transport, and decrease the metal concentration in the 2. 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