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Elements present in mineral nutrient reserves in dry Arabidopsis thaliana seeds of wild type and pho1, pho2, and man1 mutants John N.A. Lott and M. Marcia West

Abstract: Comparison of wild type and mutants of Arabidopsis thaliana offers an opportunity to study the genetic control of nutrient storage in seeds. We used energy dispersive X-ray analysis to determine the elements present and their relative amounts in globoids of dry wild-type seeds, as well as seeds of a reduced total P uptake mutant (pho1), a phosphate accumulator (pho2), and a metal accumulator (man1). Globoids are spherical inclusions, rich in phytate that function as a store for inositol, P, K, Mg, Ca, Fe, and Zn. Key findings of this study were the following: (i) globoids in protein bodies from nine different tissues and (or) organs in dry Arabidopsis thaliana seeds contained P, K, Mg, and Ca, and sometimes traces of Fe and Zn; (ii) globoids contained higher Ca and lower Mg amounts than occur in globoids in seeds of most other plant species; (iii) globoids in comparable tissue and (or) organ regions of seeds were very similar in elemental composition for wild type and all mutant plants. Key words: Arabidopsis, dry seeds, phytate, mineral nutrient mutants, phosphorus, globoids. Résumé : La comparaison du type sauvage avec des mutants de l’Arabidopsis thaliana permet d’étudier le déterminisme génétique de l’accumulation des nutriments dans les graines. Les auteurs ont utilisé l’analyse par énergie dispersive des rayons X pour déterminer les éléments présents ainsi que leurs quantités relatives dans les globoïdes de graines sèches du type sauvage, ainsi que de graines des mutants P total réduit pho1, accumulateur de phosphate pho2, et accumulateur de métal man1. Les globoïdes sont des inclusions sphériques riches en phytates qui fonctionnent comme réserve d’inositol, P, K, Mg, Ca, Fe, et Zn. Cette étude révèle que : (i) les globoïdes des corps protéiniques de neuf tissus et (ou) d’organes distincts des graines sèches de l’Arabidopsis thaliana contiennent du P, K, Mg, Ca et quelques fois des traces de Fe et de Zn; (ii) les teneurs des globoïdes sont plus élevées en Ca et plus faibles en Mg que celles des globoïdes des graines de la plupart des autres espèces; (iii) la composition en éléments chez le type sauvage et tous les mutants est tout à fait similaire pour des régions tissu et (ou) organe comparables. Mots clés : Arabidopsis, graines sèches, phytate, mutants pour nutriments, phosphore, globoïdes. [Traduit par la Rédaction]

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Introduction Arabidopsis thaliana is a widely used model plant for studies of many topics, including plant reproduction (De Bruijn et al. 1997; Robinson and Hill 1999; Girke et al. 2000; White et al. 2000). Arabidopsis offers opportunities to study mutants with genetic changes that influence uptake of mineral nutrients (Poirier et al. 1991; Delhaize et al. 1993; Delhaize and Randall 1995; Delhaize 1996; Smith et al. 1997; Dong et al. 1998). In this study we report on mineral nutrient storage particles called globoids in dry seeds of wild type and three mutants of Arabidopsis. The pho1 mutation has a reduction in total phosphate uptake into seeds to a level that is about 20% of that of the wild type (Poirier et al. 1991). The pho2 mutation is a phosphate accumulator with about a quarter more toReceived June 1, 2001. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on November 12, 2001. J.N.A. Lott1 and M.M. West. Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada. 1

Corresponding author (e-mail: [email protected]).

Can. J. Bot. 79: 1292–1296 (2001)

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tal P in seeds than the wild type (Delhaize and Randall 1995). The metal-accumulator mutant, man1, has increased seed Mn, Fe, Cu, and Zn (Delhaize 1996). The seeds of Arabidopsis contain a single layer of endosperm cells called the aleurone, and an embryo with two cotyledons and a radicle-shoot axis (Keith et al. 1994). The embryo has well-defined procambium, protoderm, and ground meristem regions in both the cotyledons and the radicle-shoot axis; and each cell contains many protein storage bodies with globoids (Mansfield and Briarty 1992). Globoids are spherical and naturally electron-dense inclusions inside protein bodies and are believed to be the location of the mineral nutrient storage compound called phytate (Greenwood and Bewley 1984; Greenwood et al. 1984; Lott 1984). Phytate is a salt of phytic acid, myo-inositol-1,2,3,4,5,6hexakis(dihydrogen phosphate) (Cosgrove 1966, 1980; IUPAC and IUPAC-IUB 1968), that commonly forms one to several percent of the embryo and (or) seed dry weight. As reviewed by Lott et al. (2000), phytate is important to the plant because it aids seedling establishment and is important to humans because of its, often negative, effects on mineral nutrient uptake in our digestive tracts. Most of the P in seeds

DOI: 10.1139/cjb-79-11-1292

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occurs as phytate. Phytate is also important in relation to crop production and sustainability because the amount of phosphorus in phytate removed with crop grains, seeds, and (or) fruits each year is large in relation to the amount of P added to crop lands as mineral fertilizer (Lott et al. 2000). Each phytic acid (PA or IP6) molecule has 12 hydrogens titratable in water on the six phosphate groups (Brown et al. 1961). The negatively charged sites on phytic acid mainly bind cations of Mg and K, but also cations of Ca, Mn, Fe, Zn, and Ba (Lott 1984; Lott et al. 1995). Studies of phytate synthesis in seeds have been reviewed by Greenwood (1989), Lásztity and Lásztity (1990), Loewus (1990), and Raboy and Gerbasi (1996). Globoids of phytate occur widely in embryo tissues, in endosperm cells that are living during germination, and in megagametophyte tissues in conifers (O’Dell et al. 1972; West and Lott 1993; Wada and Lott 1997). Our objectives in this study were as follows: (i) to provide the first comprehensive characterization of the elements P, K, Mg, Ca, Fe, Zn, and Mn that may be present in globoids in different tissues and (or) organs of wild-type Arabidopsis seeds; (ii) to compare the mean values for elements in globoids located in specific tissue and (or) organ regions of wild-type seeds with globoids from the same tissue and (or) organ regions in the pho1, pho2, and man1 mutants. This will permit identification of possible influences of these mutations on the elements that are deposited into the globoids in seeds.

Materials and methods Seeds Dry seeds of the Columbia ecotype of Arabidopsis thaliana (wild type, pho1, pho2, and man1) were kindly provided by Dr. Emmanuel Delhaize, Division of Plant Industry, CSIRO, Canberra, Australia, who had previously measured their element concentrations. The seeds were collected from plants that had been grown under the same greenhouse conditions in a potting mix with added nutrients.

Analytical electron microscopy Whole dry seeds from the three mutant lines and the wild type were prepared for microscopy using the low-water content procedure described by Lott et al. (1984). This procedure was designed to retain phytate in dry seed tissue yet permit enough expansion of the tissue for epoxy resin infiltration to occur more easily. Dry seeds were immersed in 80% ethanol for 6 h at room temperature to allow some swelling of the tissue. The seeds were then dehydrated in absolute ethanol for 22 h at room temperature, soaked in propylene oxide, infiltrated through a propylene oxide – Spurr’s resin series (over a period of 3 days), and hardened at 70°C in an oven. Electron-dense fixatives and stains were not used at any stage of sample preparation. Sections 1.0–1.5 µm thick were cut dry using an ultramicrotome. This dry-cutting method prevented extraction of water-soluble materials that can be lost in a waterfilled boat, which is routinely used in thin-sectioning (Skilnyk and Lott 1992). The thicker sections also helped to keep globoids intact, rather than being pulled out of the section or shattered as they frequently are in thinner sections. Globoids, which can be identified by their natural electron density, were examined and analyzed using a JEOL EX-II TEMSCAN scanning transmission electron microscope (JEOL USA, Inc., Peabody, Mass.) equipped with a Princeton GammaTech IMIX energy-

1293 dispersive X-ray (EDX) analysis system (Princeton Gamma-Tech, Inc., Rocky Hills, N.J.). For each of five seeds of the wild type and the pho1, pho2, and man1 mutants, five globoids from each of nine different tissue and (or) regions were examined. Thus we collected a total of 900 EDX analysis spectra. Peak-to-background ratios for P, K, Mg, Ca, Mn, Zn, and Fe were calculated using the Reid et al. (1999) background subtraction and element window width criteria. Potential overlaps of K, Fe, and Zn by Ca, Mn, and Cu, respectively, were also corrected using the Reid et al. (1999) procedures. Because of the background modeling procedure used, very small positive or negative numbers occurred when there was essentially no element present. Peak-to-background ratios are convenient for comparing globoid composition between seed types and (or) between tissue types within a given seed, but should not be considered to be quantitative measurements of element concentrations within a globoid.

Statistics MINITAB’s analysis of variance test was used to determine statistical significance of means. When differences were found in element levels, Tukey’s test was used to determine which means differed at p > 0.05. We report here statistical comparisons of selected elements in globoids of different tissues in wild-type seeds and then comparisons of each mutant line (pho1, pho2, and man1) to the wild type.

Results and discussion Globoids were found in the following nine tissues and (or) organs of dry wild type and mutant Arabidopsis seeds: aleurone; protoderm, ground meristem, and procambium of the hypocotyl-radicle axis; protoderm, ground meristem, and procambium of the cotyledons; shoot apex; radicle apex (Tables 1 and 2). In all areas globoids were naturally electrondense spheres that contained considerable P, K, Mg, and Ca; traces of Zn and Fe; and little or no Mn. Because of the importance of seeds to the survival of plants, reproductive structures generally are highly conserved. The results presented here show that the pho1, pho2, and man1 mutants had remarkably little influence on the elements present in those globoids that were made in comparable tissues. This indicates that the phytate formation and deposition process is very highly regulated and a high demand process. While our X-ray microanalysis methods reveal the presence of elements but not compounds, the presence of considerable P, K, Mg, and Ca, plus traces of Fe and Zn is consistent with the globoids in Arabidopsis seeds being made of phytate. In the wild type there were differences in the elements present in globoids formed in the nine different organ and (or) tissues studied (Table 1). For example, the P levels were lower in globoids from the aleurone and the protoderm and procambium tissues in the hypocotyl – radicle axis and cotyledons than in the ground meristem and the apices. Both the cotyledon and radicle-shoot axes showed similar trends in globoid composition as one progresses from the protoderm at the outside to the procambium at the center. From the outside towards the center the K levels rose, the Ca levels declined significantly, and Mg levels were somewhat higher in the ground meristem than in the tissues on either side. The highest Fe mean P/B ratios were in the radicle apex and the procambium regions of both the hypocotyl – radicle axis and © 2001 NRC Canada



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Table 1. Mean peak-to-background ratios for globoids of specified tissue regions in Arabidopsis thaliana wild-type seeds. Organ

Tissue

Aleurone Hypocotyl – radicle axis

Protoderm Ground meristem Procambium Protoderm Ground meristem Procambium

Cotyledon

Shoot apex Root apex

P P/B

K P/B

Mg P/B

Ca P/B

Fe P/B

Zn P/B

5.23±2.16c 5.84±1.82c 7.53±1.38a 5.09±1.78c 5.13±1.41c 6.84±1.34ab 5.50±2.09bc 6.79±1.57ab 7.60±1.66a

3.89±1.67cd 4.51±2.56bc 5.10±1.62bc 5.26±1.29bc 2.51±0.78d 4.01±1.00c 4.83±1.58bc 5.82±2.00ab 6.93±1.61a

1.83±1.32ef 2.28±0.89de 3.33±1.11bc 3.07±1.57bcd 1.25±0.52f 3.06±0.97bcd 2.56±1.19cde 4.07±1.41ab 4.61±1.24a

6.84±5.36a 8.17±3.49a 8.18±1.98a 1.76±1.44b 9.43±3.51a 7.79±2.00a 3.65±2.53b 3.54±3.55b 2.10±1.15b

0.06±0.05b 0.06 ±0.07b 0.47±0.73ab 0.63±0.79a 0.06±0.06b 0.13±0.32b 0.86±0.61a 0.13±0.25b 0.59±0.67a

0.20±0.24bc 0.44±0.44ab 0.37±0.21abc 0.40±0.21abc 0.23±0.12bc 0.18±0.14c 0.41±0.27abc 0.43±0.26ab 0.50±0.30a

Note: Within a given column, values that are followed by the same letter are not significantly different at P > 0.05. For each of the nine tissue and (or) organ regions we analyzed five globoids from each of five seeds.

Table 2. Mean peak-to-background ratios for globoids from specified tissues in Arabidopsis seeds of wild type, pho1, pho2, and man1. Tissue type

Genotype

P P/B

K P/B

Mg P/B

Ca P/B

Fe P/B

Mn P/B

Zn P/B

Aleurone

Wild type pho1 pho2 man1

5.23±2.16a 5.40±1.61a 5.01±1.56a 4.68±1.63a

3.89±1.67b 5.31±2.36a 4.59±1.82ab 3.66±1.20b

1.83±1.32b 2.64±1.10a 1.95±0.63ab 2.25±0.99ab

6.84±5.36a 6.50±2.66a 5.44±1.88a 4.90±3.78a

0.06±0.05a 0.06±0.08a 0.07±0.07a 0.07±0.04a

–0.02±0.06a –0.06±0.08a –0.04±0.06a –0.02±0.06a

0.20±0.24b 0.45±0.52a 0.25±0.23ab 0.19±0.19b

Hypocotyl – radicle axis protoderm


5.84±1.82a 5.26±1.43a 6.12±1.63a 5.67±1.51a

4.51±2.56a 3.62±0.96a 3.70±1.04a 3.63±1.56a

2.28±0.89a 1.81±0.93a 1.86±0.85a 2.43±1.01a

8.17±3.49ab 7.17±2.58ab 9.00±2.85a 6.78±1.79b

0.06±0.07a 0.07±0.07a 0.08±0.07a 0.04±0.05a

–0.03±0.07a –0.02±0.05a 0.01± 0.07a –0.04±0.07a

0.44±0.44a 0.30±0.26a 0.41±0.39a 0.57±0.65a

Hypocotyl – radicle axis ground meristem


7.53±1.38a 8.11±1.51a 7.44±1.35a 7.41±1.78a

5.10±1.62a 4.71±1.00a 5.92±1.66a 5.66±2.40a

3.33±1.11a 4.06±1.10a 3.37±0.84a 3.28±1.46a

8.18±1.98a 6.93±2.88ab 6.22±2.26b 6.34±2.29b

0.47±0.73a 0.28±0.50a 0.22±0.45a 0.38±0.89a

–0.03±0.14a –0.02±0.08a –0.03±0.05a –0.06±0.07a

0.37±0.21a 0.32±0.39a 0.18±0.21a 0.26±0.15a

Hypocotyl – radicle axis procambium


5.09±1.78b 5.61±2.07ab 6.16±1.40ab 6.41±1.44a

5.26±1.29a 3.99±1.57b 4.96±1.44ab 4.73±1.28ab

3.07±1.57a 2.81±1.42a 3.15±1.16a 3.08±1.37a

1.76±1.44c 3.79±1.34a 2.51±1.19bc 3.15±1.98ab

0.63±0.79ab 0.45±0.46b 1.00±0.81a 0.85±0.71ab

–0.08±0.07a –0.06±0.05a –0.09±0.07a –0.09±0.06a

0.40±0.21a 0.34±0.25a 0.36±0.32a 0.49±0.40a

Cotyledon protoderm


5.13±1.41a 4.71±1.49a 5.28±1.21a 4.96±1.32a

2.51±0.78bc 2.21±0.61c 3.37±0.92a 2.87±0.71ab

1.25±0.52a 1.30±0.43a 1.16±0.48a 1.46±0.60a

9.43±3.51a 9.22±3.65a 8.78±2.37a 8.97±2.47a

0.06±0.06a 0.06±0.06a 0.06±0.08a 0.05±0.06a

–0.03±0.06a 0.01±0.11a –0.01±0.06a –0.02±0.05a

0.23±0.12b 0.32±0.22b 0.42±0.32ab 0.61±0.42a

Cotyledon ground meristem


6.84±1.34b 8.07±1.49a 7.52±1.43ab 7.05±1.56ab

4.01±1.00b 4.44±1.01b 5.67±1.53a 4.63±1.13b

3.06±0.97b 4.24±1.02a 2.95±0.73b 3.44±0.98b

7.79±2.00a 8.60±3.01a 7.92±2.15a 7.14±2.12a

0.13±0.32a 0.43±0.54a 0.14±0.22a 0.39±0.59a

0.04±0.20ab –0.05±0.06b –0.01±0.09ab 0.12±0.36a

0.18±0.14ab 0.09±0.08b 0.14±0.13ab 0.17±0.08a

Cotyledon procambium


5.50±2.09a 5.58±1.54a 6.52±1.55a 5.98±1.55a

4.83±1.58a 4.44±1.65a 4.66±1.68a 4.30±1.59a

2.56±1.19b 3.53±1.38a 3.14±0.87ab 3.19±1.08ab

3.65±2.53a 2.56±1.01a 2.70±1.63a 3.85±1.58a

0.86±0.61a 0.21±0.14b 0.70±0.55a 1.02±0.78a

–0.08±0.06a –0.04±0.04a –0.05±0.06a –0.05±0.07a

0.41±0.27ab 0.35±0.20b 0.30±0.28b 0.62±0.55a

Shoot apex


6.79±1.57a 6.79±1.60a 6.83±1.47a 7.16±1.41a

5.82±2.00a 5.24±2.01a 6.43±1.62a 6.00±1.79a

4.07±1.41a 4.28±1.17a 4.00±1.20a 4.43±1.39a

3.54±3.55a 2.35±1.13ab 1.17±0.74b 1.81±1.01b

0.13±0.25b 0.46±0.63ab 0.14±0.15b 0.75±0.80a

–0.06±0.04a –0.07±0.06a –0.06±0.04a –0.09±0.06a

0.43±0.26a 0.46±0.30a 0.39±0.22a 0.53±0.23a

Root apex


7.60±1.66a 6.71±2.27a 7.38±2.15a 6.65±1.45a

6.93±1.61ab 5.50±2.23b 7.95±3.08a 5.35±2.09b

4.61±1.24a 4.14±1.99ab 3.47±1.21bc 2.85±1.42c

2.10±1.15ab 1.50±1.20b 3.35±2.94a 3.55±2.99a

0.59±0.67ab 0.93±0.88a 0.12±0.11b 0.59±0.81ab

–0.06±0.07a –0.09±0.05a –0.03±0.08a 0.02±0.29a

0.50±0.30a 0.51±0.35a 0.39±0.37a 0.35±0.33a

Note: For each tissue region, values within a column that are followed by the same letter are not significantly different at P > 0.05. For each of the nine tissue/organ regions we analyzed five globoids from each of five seeds. © 2001 NRC Canada



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the cotyledons. The globoids of wild-type seeds had traces of Zn and no Mn (data for Mn in wild-type globoids is only presented in Table 2). When globoids from seeds of pho1, pho2, and man1 were compared with the wild type for a specified organ and tissue type, the mean P/B values for P, K, Mg, Ca, Fe, Mn, and Zn were remarkably similar (Table 2). The number of differences between globoids of wild-type plants and those of the mutants was remarkably small given the large number of comparisons possible. Of the 189 possible comparisons of the wild-type globoids with globoids from the three mutants, only 11.1% were statistically different. These differences were highest with respect to the element Ca and the procambium tissue type. There was no obvious pattern in which a change in one element, such as Ca, was always matched by a reciprocal increase or decrease in another element. The man1 mutant did not result in any statistically significant increase in Mn, Fe, and Zn. With the exception of one cotyledon ground meristem globoid in a man1 seed that provided a Mn P/B ratio of 1.12, the Mn P/B values were effectively at background levels in all the tissue and (or) organ regions studied. This P/B ratio of 1.12 was more than 2 times greater than the highest Mn P/B ratio detected in any other wild-type, pho1, or pho2 globoids. Iron and Zn average P/B levels in globoids were very low and indicated that these elements are only present in trace amounts relative to P, K, Mg, and Ca. Minus P/B values reflect fluctuations around the background subtraction line. Thus slightly positive or slightly negative numbers are considered background. The pho1 mutant, with a large reduction in P, did not qualitatively show the reduction in globoid sizes and (or) numbers that we had expected. However, in the relatively thick dry-cut sections of seed tissue that we studied it was difficult to accurately determine globoid size and number. Thus we have made no attempt to quantify the volume of phytate in globoids. We have no evidence relating the density of the packaging of elements in different globoids. EDX analysis determines the relative proportions of different elements in the sample area irradiated by the electron beam but it does not measure the density of the packaging of those elements. In other words, the amounts of P, K, and Mg could be in similar proportions to each other in globoids that differed in absolute density. We observed that Ca in Arabidopsis globoids was higher than has generally been found in globoids from other plant species where Ca is either lacking or present in trace amounts. Not only was this true for Ca P/B ratios, but the Ca:P ratios (ranging from 0.27–1.82 in wild-type seeds) were also much higher than generally found in globoids in seeds of other plant species, where Ca:P ratios seldom reach 0.1. Seed size is likely not a factor, since small Begonia seeds (approximately 70 000 seeds/gram (Harthun 1975)) have little if any Ca in globoids (West and Lott 1991) compared with Arabidopsis seeds, which are also small (approximately 50 000 seeds per gram (Meyerowitz 1987)). Most seed embryos have comparatively small Ca concentrations; the major exception being those species that contain calcium oxalate crystals in certain protein bodies (Spitzer and Lott 1982). Experimental attempts to modify the mineral nutrients in protein bodies through the addition of K, Ca, or Mg salts to developing seeds also showed that the developing

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seed is very resistant to alteration of the mineral nutrients stored in globoids (Lott et al. 1985; Lott et al. 1994). Some key findings from this research are the following: (i) protein bodies from nine different tissue and (or) organ regions in dry Arabidopsis seeds contained spherical globoids that were rich in P, K, Mg, and Ca and also sometimes contained traces of Fe and Zn; (ii) the globoids contained higher Ca than generally occurs in globoids from seeds of other plants that have been studied; (iii) globoids from the shoot and root apices had the lowest Ca and the highest Mg levels of globoids in the nine tissues and (or) regions studied; (iv) globoids from the mutants, pho1, pho2, and man1, for a given tissue and (or) organ region were very similar to wild-type globoids.

Acknowledgments The authors thank Dr. E. Delhaize, CSIRO, Division of Plant Industry, Canberra, Australia for providing seeds that made this study possible. We also thank Dr. Irene Ockenden and Mr. Klaus Schultes for their advice and help. This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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