Trace Element and Rare Earth Element Profiles in Berry Tissues of Three Grape Cultivars

Trace Element and Rare Earth Element Profiles in Berry Tissues of Three Grape Cultivars Yumei Yang, 1 Changqing Duan, 2 Huijuan Du, 1 Juanjuan Tian, 1 and Qiuhong Pan 3 * Abstract: Mineral elements play an important role in the geographic traceability of wine, and understanding mineral element profiles in berry tissues helps to determine the relationship between elemental accumulation and regional flavor formation in grapes and wine. Eighteen trace elements and 15 rare earth elements were investigated in the skin, pulp, and seeds of grape cultivars Cabernet Sauvignon, Marselan, and Italian Riesling using inductively coupled plasma mass spectrometry (ICP MS). Most trace elements, except for B, Zr, Tl, and U, presented similar tissue-specific distributions in their concentrations in the order of seeds > skin > pulp, but concentrations varied within the three cultivars. The two red cultivars showed significantly higher concentrations of Cu, Cr, Ba, Mo, Cd, Ga, Ge, and Tl and lower concentrations of B, Mn, Sr, and U than the white cultivar Italian Riesling. However, the tissue distribution of rare earth elements was totally different from that of the trace elements. In Italian Riesling berries, concentrations of most rare earth elements in the skin were greater than or similar to those in the seeds and pulp, except that Yb was not detected. For the two red cultivars, concentrations of 11 rare earth elements (Y, La, Ce, Nd, Pr, Sm, Gd, Dy, Ho, Er, and Yb) in the seeds were not as high as those in the skin and pulp. These findings give new insights into the tissue distribution of trace and rare earth elements in white and red grape cultivars. Key words: trace elements, rare earth elements, berry tissues, grape cultivars Although plants contain only microscopic or trace levels of trace elements and rare earth elements (REEs), these elements play important roles in regulating their growth and development. Such elements as boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn) are involved in many metabolic and cellular functions, including enzyme activation and protein stabilization, and are thus considered essential to all higher plants (Hänsch and Mendel 2009). They also influence the yield and quality of fruit. Fe deficiency can cause leaf chlorosis, which leads to a decrease in photosynthetic efficiency (Veliksar et al. 1995, Morales et al. 2000), and the application of Zn and B to soils deficient in them can significantly increase the yield of fruit and the level of protein and 17 types of amino acids in pear fruit (Shi and Cheng 2004). In grape berries, Zn improves the retention of bunches onto the branches and B affects the number and size of berries (Da Silva et al. 2008). At correct concentrations, REEs 1 Postgraduate Student, 2 Professor, and 3 Associate Professor, Center for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. *Corresponding author (email: panqh@cau.edu.cn; fax: 86-10-62737136) Acknowledgments: This work was supported by the National Natural Science Foundation of China (grant 30771490 to Pan Q.H.) and the Earmarked Fund for the Modern Agro-Industry Technology Research System (nycytx-30). Supplemental data is freely available with the online version of this article. Manuscript submitted Nov 2009, revised Mar 2010, accepted Apr 2010. Publication costs of this article defrayed in part by page fees. Copyright 2010 by the American Society for Enology and Viticulture. All rights reserved. also show positive effects on photosynthetic reactions and the growth of fruit (Wahid et al. 2000, Liu et al. 2009). This group of elements includes yttrium (Y) and scandium (Sc) and 15 lanthanides: lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). In general, La, Ce, Pr, Nd, Sm, and Eu are light rare earth elements (LREEs) and Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Y are heavy rare earth elements (HREEs) (Tyler 2004). Previous studies indicate that REEs improve the biosynthesis of secondary metabolites such as flavonoids and phenylethanoid glycosides (Ouyang et al. 2003, Yang et al. 2005, Ge et al. 2006), which suggests that REEs might be related to the formation of flavor attributes in wines. In addition, REEs are found to affect the absorption of other elements. It has been reported that these elements inhibit iron deficiency in cucumber (Johnson and Barton 2007); also, as the radii of REE ion is close to that of Ca 2+, they affect Ca 2+ uptake and replacement and interfere with other physiological functions of Ca 2+ (Thompson Amann et al. 1992, Cheng et al. 1999, Zhou and Zhang 2000, Diniz and Volesky 2005). The mineral nutrient status of the grape berry is of concern to both viticulturists and enologists because it has a direct impact on berry nutrition, juice, and must composition. According to these previous reports, we speculate that both trace elements and REEs might be involved, directly or indirectly, in the regulation of the biosynthetic metabolisms of some flavor compounds, ultimately affecting fruit quality and yield. However, the mechanism by which these elements regulate flavor metabolism remains unclear. 401

402 Yang et al. Recent research has examined the accumulation and tissue distribution of mineral elements in grape berries. One study analyzed the transportation patterns of 10 common mineral elements calcium (Ca), potassium (K), phosphorus (P), magnesium (Mg), sulfur (S), Mn, Fe, B, Cu, and Zn from vascular flow into the berries, but did not address REE accumulation patterns (Rogiers et al. 2006). In a study on the distribution of 13 REEs in Chardonnay berries, 57% of REEs were localized in the skin, 40% in the flesh, and 3% in the seeds; for europium (Eu), however, its content in the seeds was ~20% higher than that in the skin and the flesh (Bertoldi et al. 2009). Grapes at different developmental stages may acquire mineral nutrients differently. During the veraison-to-ripeness stage, sugar content rapidly increases, acids sharply decrease, and secondary products such as aromatic compounds and polyphenolics also accumulate significantly (Ollat et al. 2002). These flavor compounds accumulate in different tissues of berries. The skin generally provides volatile, nonvolatile, and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of the sensory characteristics of wine. Key questions include what changes take place in the distribution of trace elements and REEs in different berry tissues from veraison to ripeness and whether there are differences between red and white grape cultivars. Answers to these questions may help to clarify the elemental impact on the formation of flavor quality. Thus, the aims of this study are to compare the distribution of mineral elements in various tissues and different grape cultivars and their changes after veraison and to provide a basis for further studies concerning the relationship between mineral elements and the formation of fruit quality. Materials and Methods Sampling. Two red cultivars, Cabernet Sauvignon and Marselan, and one white cultivar, Italian Riesling, were regularly sampled during 2007 from the end of veraison to commercial berry harvest. There were eight samplings of Cabernet Sauvignon and Marselan, and five samplings of Italian Riesling. These cultivars had been grown for nearly 10 years in adjacent vineyards in the Qilian winemaking region (lat. 39.14 N; long. 99.84 E), Gansu Province, China. These three vineyards were chosen because of similar soil quality (gravel soil). The Cabernet Sauvignon vineyard is located on the eastern edge of the Marselan vineyard and the northern edge of the Italian Riesling vineyard. These vineyards are separated by a distance of ~50 m. Each vineyard occupies an area of ~4.0 ha and the planting distances are 2.5 1 m. These vineyards have almost identical environmental factors, such as soil characteristics and climate, and underwent the same management practices during the experimental period, including irrigation, fertilization, soil management, disease control, and pruning. The length of time required for the grape berries to begin veraison varied. At each stage, two 100-berry samples of each cultivar were selected from at least five 10-cluster selections at similar positions on 50 whole-vine selections. The skin, pulp, and seeds were separated by hand immediately, frozen in liquid nitrogen, ground to a powder, and stored at -80 C until use. Sample digestion. The pulp, skin, and seed powder were weighed to ~1.0000 g, 0.5000 g, and 0.2000 g, respectively, directly into poly(tetrafluoroethylene) (PTFE) vessels. Five ml of HNO 3 (65%, v/v, mass spectrometry [MS] grade; Merck, Darmstadt, Germany) and 0.25 ml hydrofluoric acid (40%, v/v, MS grade; Merck) were added to the PTFE vessels. Then the vessels were placed on an electric heating board (DRB-3; Ounuo Ltd., Tianjin, China) and heated at 150 C, for 1.5 hr (for pulp and skin) or for 2 hr (for seeds). During the digestion period, the vessels were gently shaken by hand at intervals to keep the samples heating uniformly. Once the solid in the vessels disappeared and the solution became clear, 1 ml H 2 O 2 (30%, v/v, MS grade; Merck) was added to the vessels, which were gently shaken for 45 min. After complete digestion, the PTFE vessels were cooled to room temperature, and the digested solutions were transferred to 50-mL polyethylene terepthalate (PET) vials and the PTFE vessels were rinsed at least three times with ultrapure water. The rinse water was also pooled into the PET vials, and then ultrapure water was added to the solutions until they weighed 40 g. To determine the concentration of elements in the pulp, skin, and seeds from each stage, each tissue sample was digested in at least three independent replicates and each replicate was analyzed three times. Analysis by inductively coupled plasma mass spectrometry (ICP MS). The trace element calibration solution included Mn, Fe, Cu, Zn, chromium (Cr), barium (Ba), Mo, cadmium (Cd), cobalt (Co), Ni, thallium (Tl), and uranium (U). Iron was diluted by gradient to 5000, 2000, 500.0, 100.0, and 20.00 μg/l with HNO 3 (5%, v/v, MS grade; Merck) and the others to 50.00, 20.00, 5.000, 1.000, and 0.200 μg/l with HNO 3 (5%, v/v, MS grade; Merck). The rare earth element calibration solutions (Agilent Technologies, Santa Clara, CA) included La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Y were gradiently diluted to 0.4000, 0.1000, 0.0200, 0.0050, and 0.0010 μg/l with HNO 3 (5%, v/v, MS grade; Merck). The individual calibration standard solutions B, germanium (Ge), and strontium (Sr) (CISRI, Beijing, China) were mixed and diluted to 2.000, 0.5000, 0.1000, 0.0500 μg/l with ultrapure water. The individual calibration standard solutions zirconium (Zr), palladium (Pd), and gallium (Ga) (CISRI) were diluted to 2.000, 0.5000, 0.1000, 0.0500 μg/l with HCl (10% v/v, MS grade; Beijing Chemical Technology Institute, China). Rhodium (Rh) (CISRI) with a concentration of 1 mg/l was chosen as the internal standard element. A quadrupole-based ICP MS (7500 a; Agilent Technologies) with a Babington nebulizer was used in sample analyses. Sample introduction was performed via a peristaltic pump. The instrument was tuned with a 10 μg/l tuning solution of 7 Li (lithium), Y, Ce, and Tl. Their relative standard deviations (RSD) were all <3%. The CeO/ Ce ratio was set below 0.5% and the Ce 2+ /Ce + ratio was <2.0%. Instrument operating parameters were as follows:

Trace and Rare Earth Elements in Berry Tissues 403 RF power, 1,300 W; plasma gas flow, 15 L/min; carrier gas flow, 1.15 L/min; acquired depth, 7.2 mm; dwell time (main group elements), 0.1 sec per point, 3 points per mass; resolution, 0.65 0.80 amu at 10% peak height (Du et al. 2009). Analysis of method availability. For verification of the availability of the method, it is necessary to show the limit of detection (LOD), limit of quantitation (LOQ), and relative standard deviation (RSD) of each element. As the procedures for elemental determination for berry skin, pulp, and seeds were the same apart from digestion times (1.5 hr for skin and pulp, 2 hr for seeds) a 2-hr digestion time was used to digest whole berry samples (including skin, pulp, and seeds) and other steps were carried out as described above. Under these conditions, we used 11 independent blank solutions of 5% HNO 3 for trace elements and three independent standard solutions containing various elements of 10 ng /ml for REEs. LOD and LOQ were calculated with the formulas: LOD (ng/ml) = [3σ /(S-B)] C LOQ = [10σ /(S-B)] C where σ represents the standard deviation for the blank solution; S represents the ICP-MS signal strength of each element at 10 ng/ml; B represents the ICP-MS signal strength of the blank solution; and C represents the concentration of each standard element. RSD was shown by the percentage of the standard deviation from the mean concentration of each element in the berry samples. The berry samples were divided into six parts, and each part was analyzed using independent digestion and ICP MS. A total of 18 results were used for the calculation of RSD. Except for Gd (7.81%) and Dy (8.76%), RSDs were <5%, indicating that the method used in this study produced precise results with excellent reproducibility. Data analysis. Each data point used for mapping was from an average of nine replications. These data were mapped using a SigmaPlot 10.0 box plot (Systat Software, San Jose, CA), and each box plot was calculated from eight data points (for Cabernet Sauvignon and Marselan) and five data points (for Italian Riesling) to display the element range and median. Results Distribution of trace elements. Eighteen trace elements were detected in the pulp, skin, and seeds of the three grape cultivars (Figure 1; Supplemental Tables 1 9), and concentrations of all trace elements were much higher than their LOQ (Table 1). The trace elements were divided into three groups according to concentrations: (1) a few to tens of micrograms; (2) tens to hundreds of nanograms; and (3) a few to tens of nanograms. Fe had the highest levels in all the grape berry tissues, and Sr, Mn, Zn, B, and Cu had the next highest, all at microgram levels. The second-level elements were Cr, Ba, Ni, and Mo, and the third-level elements were Ga, Zr, Pd, Co, Ge, Cd, Tl, and U. Concentrations of most trace elements, such as Mn, Fe, Cu, Zn, Sr, Ba, Mo, Pd, Cd, Ga, and Ge, in the seeds were very significantly higher than in the skin and pulp, whereas concentrations in the skin were generally higher than in the pulp. However, there were some exceptions. For example, red cultivars contained similar B concentrations in all three berry tissues and similar Co concentrations in skin and seeds. In the white cultivar, B concentration was higher in the skin than in the pulp and seeds, while Co concentration was in the sequence seeds > skin > pulp. Tl concentrations were similar among the three tissues of the same cultivars but higher in the red grape cultivars than in the white cultivar. U concentrations increased in the sequence skin > seeds > pulp and varied greatly in skins of the two red grape cultivars. In the white cultivar, Zr concentration was similar in the skin and seeds and higher than in the pulp. In the red cultivars, Zr concentration was highest in the skin, second in the pulp, and lowest in the seeds. There were great differences in concentrations of some trace elements, such as Sr, Ba, Mo, Cd, Ga, and Ge, between the red cultivars and the white cultivar, while concentrations of other trace elements were only slightly different. In particular, Sr concentration was more than four times higher in white cultivar seeds than in red cultivar seeds. However, for most trace elements, there were no significant differences in concentrations between Cabernet Sauvignon and Marselan. These results indicate that the distribution of trace elements was tissue dependent yet varied to a certain extent from the red to white cultivars. In addition, some elements such as Cr in Cabernet Sauvignon skin and Ni in Cabernet Sauvignon pulp and Italian Riesling seeds showed great variations from the end of veraison to harvest. Distribution of rare earth elements. The distribution pattern of 15 REEs in the various tissues of the grape berries was different than that of trace elements and largely depended on grape cultivar (Figure 2; Supplemental Tables 10 18). In the white cultivar, the skin tissue contained the highest levels of 12 of the 15 REEs. These 12 REEs were also present in higher or similar concentrations in the pulp than in the seeds. Yb was not detected in the skin (<LOQ, 15.803 ng/kg; Table 2), whereas Eu and Lu concentrations were similar in the skin and seeds. There were also differences in the distribution of REEs in the two red cultivars. In Cabernet Sauvignon, most REEs, such as La, Ce, Nd, Pr, Sm, Gd, Tb, Dy, Ho, Er, and Tm, had similar concentrations in the pulp and skin but were lowest in the seeds, whereas the concentration of some elements, such as Yb and Lu, were highest in the pulp and second highest in the skin. There were some exceptions; for example, Eu concentration was highest in Cabernet Sauvignon seeds. REE distribution in various berry tissues was slightly different in Marselan than in Cabernet Sauvignon. The distribution of some REEs, such as Y, La, Ce, Nd, Pr, Sm, Gd, Dy, Er, and Yb, was similar to that in Italian Riesling, the concentrations of which were highest in pulp and seed. Sm was not detected in Marselan seeds and there was no Lu in the pulp. Eu, Tb, Tm, and Lu concentrations were higher in Marselan seeds than in pulp and skin. The

404 Yang et al. Figure 1 Concentrations of 18 trace elements in pulp, skin, and seeds of three Vitis vinifera cultivars. Each box plot was composed of eight data points for Cabernet Sauvignon (CS) and Marselan (M) and five data points for Italian Riesling (IR). The median of the data is indicated by a horizontal line in the interior of the box. The height of the box is equal to the interquartile distance (the difference between the third and the first quartile).

Trace and Rare Earth Elements in Berry Tissues 405 Figure 2 Concentrations of 15 rare earth elements in the three tissues of three Vitis vinifera cultivars. Each box plot was composed of eight data points for Cabernet Sauvignon (CS) and Marselan (M) and five data points for Italian Riesling (IR). The median of the data is indicated by a horizontal line in the interior of the box. The height of the box is equal to the interquartile distance (the difference between the third and the first quartile). distribution of Eu was largely different than that of the other 15 REEs; Eu concentration increased from the seeds to the skin to the pulp in all three grape cultivars, which illustrates Eu is a positive anomaly in seeds. Discussion In terms of the distribution of trace elements and REEs in various berry tissues, similar results have been seen, over the past few years, in other reports. In Shiraz berries, the trace elements Mn, Fe, Cu, and Zn presented similar tissue-specific distribution with seeds > skin > pulp (Rogiers et al. 2006), while in Chardonnay tissues concentrations of most REEs were in the order skin > pulp > seeds (Bertoldi et al. 2009). Moreover, the REE Eu was detected at a higher concentration in the seeds of the three cultivars studied here, which is similar to a study that suggested that seeds selectively absorb Eu (Bertoldi et al. 2009). However, the earlier studies reported that Cu, Fe, Mn, and Zn concentrations were not significantly different in the pulp, skin, and seeds of the grape cultivar Tempranillo (Esparza et al. 2004), perhaps because the elemental concentrations were evaluated on the basis of dry tissue weight, whereas our study was on a fresh weight basis. In fresh ripening berries, pulp contains much larger amounts of water than the

406 Yang et al. Table 1 Limit of detection (LOD), limit of quantitation (LOQ), and relative standard deviation(rsd) for 18 trace elements under study. Element LOD LOQ RSD (%) B 0.079 0.263 1.97 Mn 0.081 0.270 0.80 Fe 0.017 0.057 1.31 Cu 0.059 0.197 1.01 Zn 1.752 5.840 1.03 Cr 7.894 26.313 0.98 Sr 0.009 0.030 1.98 Ba 1.310 4.367 1.96 Mo 3.514 11.713 3.39 Zr 1.208 4.027 3.24 Pd 3.180 10.600 1.86 Cd 8.718 29.060 1.72 Co 0.477 1.590 2.52 Ni 1.309 4.363 1.01 Ga 0.417 1.390 0.46 Ge 2.422 8.073 4.55 Tl 3.484 11.613 3.76 U 0.569 1.897 0.33 Table 2 Limit of detection (LOD), limit of quantitation (LOQ), and relative standard deviation (RSD) for 15 rare earth elements under the present experimental conditions. Element LOD LOQ RSD (%) Y 0.142 0.473 0.48 La 0.861 2.870 0.81 Ce 0.743 2.477 0.51 Pr 1.419 4.730 3.16 Nd 2.486 8.287 3.62 Sm 1.931 6.437 0.31 Eu 4.648 15.493 4.92 Gd 15.340 51.133 7.81 Tb 3.201 10.670 3.02 Dy 8.369 27.897 8.76 Ho 2.239 7.463 4.78 Er 7.753 25.843 3.47 Tm 2.195 7.317 2.43 Yb 4.740 15.800 1.09 Lu 3.015 10.050 4.49 skin or seeds. However, since most REEs are more highly concentrated in the skin and pulp than in the seeds, a difference in the water content should not cause differences in elemental concentrations in berry tissues. A tissue-dependent distribution of trace elements and REEs may be related to the biological roles of the elements in plant tissues. For example, B plays a predominant role in the formation of primary cell walls, at it cross-links pectic polysaccharide rhamnogalacturonan II (RG-II) to form B-RG-II (O Neill et al. 1996, Matoh 1997), and RG-II was found mainly in the skin tissue of grape berries (Vidal et al. 2001), which corresponds to our result that there was more B in the skin than in the pulp. Many studies have indicated that trace elements can be used to fingerprint regional wines (Taylor et al. 2003, Coetzee et al. 2005, Angus et al. 2006, Galgano et al. 2008), in agreement with our results indicating that three cultivars from the same growing environment also show similar trace element distribution. In general, the winemaking process for red and white wine is different; the former is commonly made by fermenting crushed berries, whereas the latter is usually made from fermenting grape juice. Since REEs are mainly found in skin and pulp, red wines should have higher concentrations of REEs than white wines (Mihucz et al. 2006). Trace elements present similar tissuedependent distribution, and red and white cultivars show great differences in the concentrations of some of these elements. In addition, grape cultivars show great differences in REE concentrations, thus posing some questions about the role of trace elements and REEs in the formation of grape varietal flavor and whether REEs contribute more to the formation of varietal flavor than do trace elements. To answer these questions, we will need to determine the effect of various trace elements and REEs on the formation of aromatic compounds and other quality parameters that determine the f lavor of wines. Conclusion The present study indicates that trace elements are generally present in their highest levels in seeds and their lowest levels in pulp, whereas most REEs are present in their highest levels in skin tissue. Also trace element profiles in all three parts of the berries show only minor differences between grape cultivars, while REEs profiles were significantly different. Literature Cited Angus, N.S., T.J. O Keeffe, K.R. Stuart, and G.M. Miskelly. 2006. Regional classification of New Zealand red wines using inductivelycoupled plasma-mass spectrometry (ICP-MS). Aust. J. Grape Wine Res. 12:170-176. Bertoldi, D., R. Larcher, G. Nicolini, M. Bertamini, and G. Concheri. 2009. Distribution of rare earth elements in Vitis vinifera L. Chardonnay berries. Vitis 48:49-51. Cheng, Y., H.Y. Yao, H.K. Lin, J.F. Lu, R.C. Li, and K. Wang. 1999. The events relating to lanthanide ions enhanced permeability of human erythrocyte membrane: Binding, conformational change, phase transition, perforation and ion transport. Chem.-Biol. Interact. 121:267-289. Coetzee, P.P, F.E. Steffens, R.J. Eiselen, O.P. Augustyn, L. Balcaen, and F. Vanhaecke. 2005. Multi-element analysis of South African wines by ICP-MS and their classification according to geographical origin. J. Agric. Food Chem. 53:5060-5066. Da Silva, M.A.G., M.A. Pavan, A.S. Muniz, T.A. Tonin, and T. Pelizer. 2008. Nutrient availability in the soil and its absorption, transport, and redistribution in vines. Commun. Soil Sci. Plant Anal. 39:1507-1516. Diniz, V., and B. Volesky. 2005. Biosorption of La, Eu and Yb using Sargassum biomass. Water Res. 39:239-247. Du, H.J., J.J. Tian, C.Q. Duan, and Q.H. Pan. 2009. Determination of 56 elements in grapes by inductively coupled plasma mass spectrometry (ICP-MS) with electric heating board digestion. Chin. J. Anal. Lab. 28:294-298. Esparza, I., I. Salinas, I. Caballero, C. Santamaría, I. Calvo, J.M. García-Mina, and J.M. Fernández. 2004. Evolution of metal and

Trace and Rare Earth Elements in Berry Tissues 407 polyphenol content over a 1-year period of vinification: Sample fractionation and correlation between metals and anthocyanins. Anal. Chim. Acta 524:215-224. Galgano, F., F. Favati, M. Caruso, T. Scarpa, and A. Palma. 2008. Analysis of trace elements in southern Italian wines and their classification according to provenance. Food Sci. Technol. 41: 1808-1815. Ge, F., X.D. Wang, B. Zhao, and Y.C. Wang. 2006. Effects of rare earth elements on the growth of Arnebia euchroma cells and the biosynthesis of shikonin. Plant Growth Regul. 48:283-290. Hänsch, R., and R.R. Mendel. 2009. Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Cur. Opin. Plant Biol. 12:259-266. Johnson, G.V., and L.L. Barton. 2007. Inhibition of iron deficiency stress response in cucumber by rare earth elements. Plant Physiol. Biochem. 45:302-308. Liu, X., H. Huang, C. Liu, M. Zhou, and F. Hong. 2009. Physicochemical property of rare earths Effects on the energy regulation of Photosystem II in Arabidopsis thaliana. Biol. Trace Elem. Res. 130:141-151. Matoh, T. 1997. Boron in plant cell walls. Plant Soil 193:59-70. Mihucz, V.G., C.J. Done, E. Tatár, I. Virág, G. Záray, and E.G. Baiulescu. 2006. Influence of different bentonites on the rare earth element concentrations of clarified Romanian wines. Talanta 70: 984-990. Morales, F., R. Belkhodja, A. Abadía, and J. Abad. 2000. Energy dissipation in the leaves of Fe-deficient pear trees grown in the field. J. Plant Nutr. 23:1709-1716. Ollat, N., P. Diakou-Verdin, J.P. Carde, F. Barrieu, J.P. Gaudillère, and A. Moing. 2002. Grape berry development: A review. J. Int. Sci. Vigne Vin 36:109-131. O Neill, M.A., D. Warrenfeltz, K. Kates, P. Pellerin, T. Doco, A.G. Darvill, and P. Albersheim. 1996. Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. J. Biol. Chem. 271:22923-22930. Ouyang, J., X.D. Wang, B. Zhao, X.F. Yuan, and Y.C. Wang. 2003. Effects of rare earth elements on the growth of Cistanche deserticola cells and the production of phenylethanoid glycosides. J. Biotechnol. 102:129-134. Rogiers, S.Y., D.H. Greer, J.M. Hatfield, B.A. Orchard, and M. Keller. 2006. Mineral sinks within ripening grapes berries (Vitis vinifera L.). Vitis 45:115-123. Shi, M., and R. Cheng. 2004. Effects of zinc and boron nutrition on balsam pear (Momordica charantia) yield and quality, and polyamines, hormone, and senescence of its leaves. Chin. J. Appl. Ecol. 15:77-80. Taylor, V.F., H.P. Longerich, and J.D. Greenough. 2003. Multielement analysis of Canadian wines by inductively coupled plasma mass spectrometry (ICP-MS) and multivariate statistics. J. Agric. Food Chem. 51:856-860. Thompson Amann, B., P. Mulqueen, and W.D. Horrocks, Jr. 1992. A continuous spectrophotometric assay for the activation of plant NAD kinase by calmodulin, calcium(ii), and europium(iii) ions. J. Biochem. Biophys. Methods 25:207-217. Tyler, G. Rare earth elements in soil and plant systems A review. 2004. Plant Soil 267:191-206. Veliksar, S.G., R.F. Syrcu, V.M. Busuioc, S.I. Toma, and A.I. Zemshman. 1995. Iron content in grape tissue when supplied with ironcontaining compounds. J. Plant Nutr. 18:117-125. Vidal, S., P. Williams, M.A. O Neill, and P. Pellerin. 2001. Polysaccharides from grape berry cell wall. Part 1: Tissue distribution and structural characterization of the pectic polysaccharides. Carbohydr. Polym. 45:315-323. Wahid, P.A., M.S. Valiathan, N.V. Kamalam, J.T. Eapen, S. Vijayalakshmi, R.K. Prabhu, and T.R. Mahalingam. 2000. Effect of rare earth elements on growth and nutrition of coconut palm and root competition for these elements between the palm and Calotropis gigantean. J. Plant Nutr. 23:329-338. Yang, S., X. Liu, D. Guo, and J. Zheng. 2005. Effect of rare-earth element Eu 3+ on callus growth and flavonoids content in Glycyrrhiza uralensis. J. Chin. Med. Mater. 28:533-534. Zhou, X.B., and W.Y. Zhang. 2000. Effect of Nd (NO 3 ) 3 on membrane damage and peroxidization process in rape during Ca 2+ starvation. J. Chin. Rare Earth Soc. 18:63-66.