Brooks, R.R.

Jaffré, Tanguy

Lee, J.

Reeves, R.D.

Horizon / Pleins textes

Phyfochemisfry, 1977, Vol. 16, pp. 1503-1505. Pergamo" Press. Printed in England. 
't 
ISOLATION AND IDENTIFICATION OF A CITRATO-COMPLEX 
OF NICKEL FROM NICKEL-ACCUMULATING PLANTS 
JULIAN LEE*, ROGER D.  REEVES*, ROBERT R. BROOKS* and TANGUY JAFFRÉ? 
*Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, New Zealand; 
?Centre O.R.S.T.O.M., Nouméa, New Caledonia 
(Received 3 March 1977) 
Key Word Index-Sebertia acuminata; Sapotaceae; Hoinaliuin species; Flacourtiaceae; nickel-accumulating 
plants ; citratd-nickelate complex. 
Abstract-An organometallic complex of nickel has been isolated from nickel-accumulating plants of New Caledonia 
and has been identified by a combination of infrared spectroscopy, high-voltage paper electrophoresis, gas-liquid 
chromatography and mass spectrometry. Most of the nickel in Sebertia,acumiilata, Homaliuriifiancii, Hoinaliurn 
guillainii, Hoinaliuin kai?aliense, Hybaiathus austrocaledonicus and Hybaizthus caledoizicus is found as a negatively- 
charged citratonickelate (II) complex with Ni(H,O); 'as the major cationic constituent. 
INTRODUCTION 
The uptake and translocation of elements such as nickel 
by plants is of considerable interest, and the need for 
efforts to identify carriers of this and other elements in 
xylem fluids has been noted [I]. Certain plant species can 
accumulate remarkably high concentrations of one or 
more of these elements, up to 10-25 % (dry-weight basis) 
in the extreme case ofthe nickel content ofthe New Caledo- 
nian species Sebertia acuminata Pierre ex Baill. The pre- 
sence of such high concentrations raises interesting ques- 
tions in plant chemistry and is a major factor stimulating 
research into the chemical form of transition elements in 
plants. Much of the existing information has come from 
electrophoretic experiments using radioactive tracers 
because of the generally low physiological levels of these 
elements. Unidentified anionic complexes of nickel and 
copper occur in xylem exudates of plants such as tomato, 
cucumber and peanut [2]. Anionic forms of copper and 
zinc have been found in ryegrass extracts [3] and much 
of the zinc in extracts of Ricinus comiiiuitis is bound to  
anionic organic complexes, possibly containing phos- 
phorus [4]. Tiffin [l, 51 found that iron (III) in several 
plhnt exudates showed the same electrophoretic be- 
haviour as anionic iron (111)-citrate complexes, but 
suggested [l, 61 that amino acids act as carriers for 
copper and nickel, as these elements show a particular 
affinity for nitrogen-containing ligands.. Lyon et al. 
[7] identified three anionic complexes of chromium in 
Leptosperinuin scoparium of which the major one was 
trioxalatochromium (III). Farago et al. [SI, using paper 
chromatography with ammonium oxalate extracts of 
Hybanthus Jloribuizdus, showed the .presence of a nickel 
compound with R ,  similar to  that of nickel pectinate 
but also noted the possibility that nickel was associated 
with amino acids. However no association between 
nickel and amino acids was found by Kelly et al. [9] 
in their study of Hybanthus species from New Caledonia. 
Severne [lo] studied the composition of nickel com- 
plexes in H. Jloribundus and although he was not able to  
4 
>- s 
isolate a pure complex, he deduced that nickel was in the 
form of a complex of low molecular weight. Perhaps the 
most significant advances to date in the elucidation of the 
structure of nickel complexes in accumulator plants are 
to be found in the work of Pelosi et al. [ l l ,  121 who studied 
extracts of Alyssum bertolonii Desv. and deduced that 
nickel was complexed with organic acids such as malic 
and malonic acids. 
In the present work, we report the results of studies 
carried out on the nature of nickel complexes extracted 
from six species of accumulator plants from New Cale- 
donia, all of which normally contain nickel concentra- 
tions in their leaves and latex, well in excess of 1000 yg/g 
(0.1 %) expressed on a dry-weight basis. The following 
were studied : Sebertia acuiniizata [ 131, Hybaial'kus 
austrocaledonicus Schinz et Guillaumin, Hybanthus cale- 
donicus (Turcz.) Cretz., Homaliuni fraiicii Guillaumin, 
Hoinaliuin guillainii Briq., and Honialium kanaliense Briq. 
Hyperaccumulation of nickel by most of these species has 
been reported by Brooks et al. [14]. In each case the 
complexes were purified by chromatography on Sephadex 
G-10 or G-25. 
IDENTIFICATION OF NICKEL COMPLEXES 
U 
The IR spectra of a solution of the nickel complex 
showed in every case, carboxylate stretching frequencies 
at 1590cm-' and 1430cm-l as well as frequencies 
characteristic of H,O bending and OH stretching 
vibrations. The MS of the effluent from the gas chromato- 
graph of the TMS derivatives of the complex showed the 
presence of a single peak with only traces of impurities. 
The spectrum showed a peak of highest mass at m/e = 
465, derived from a molecular ion of mass 480 (the 
molecular ion peak is often entirely absent from spectra 
of hydroxyacid TMS derivatives [l5] from which the 
largest detectable fragment is that formed by loss of 
-Me). Accurate measurement of the masses of several 
fragments led to  a mass of 480.185 and molecular formula 
CI8H4,,Q7Si4 for t h e i ~ M ~ a ~ e ~ ~ a ~ y ~ ~ ~ r ~ ë ~ p ~ n d i n ~ ~ Q ~  
m.1 d Y &Lb* 5977 
l,", 
1504 J. LEE et d. 
S. acuminato extract 
c (4 
O 
t 
.- 
L + H. guilloinii extract 
Ni citrate I 
Distance, cm 
Fig. 1. Electrophoretic distribution of nickel in extracts of 
S. acuminata (O) and H. guillainii (O), compared with Ni(H,O)g' 
(A) and a 2: 1 citrate:nickel solution a). Pyridine/acetic 
acid/water at pH 6.5, 3 kV, 40 min, Whatman No. 1 paper. 
a parent compound C,H80,. Known compounds of this 
formula include the saccharic acid monolactones, citric 
acid and isocitric acid. GLC of TMS derivatives of 
monolactones of galactaric acid and glucaric acid, using 
OV-101, gave retention times approximately twice those 
of the unknown TMS derivative, which was inseparable 
from derivatives of citric and isocitric acids on both 
OV-101 and OV-17. However, the MS clearly showed the 
presence of citrate in the nickel-containing plant extracts. 
The major peaks in the spectrum of silylated citric acid 
correspond to those reported previously [16]. 
Experiments with high-voltage paper electrophoresis 
(Fig. 1) show the behaviour of the nickel at p H  6.5 in 
two of the plant extracts, compared with Ni(H,O);+ 
and with nickel in a 2 : l  citrate/nickel solution. The 
combined evidence of GLC and electrophoresis showed 
the presence of an anionic citrato-complex of nickel in 
the purified extracts of all the nickel-containing plants 
noted above. Varying amounts of Ni(h,O)z+-were also 
present. Mass spectrometric confirmation of the identity 
of the citrate was carried out for the extracts from 
S. acuminata and H. austrocaledonicus. 
The electrophoretic behaviour and the elemental 
analysis of the plant extracts are consistent with the 
presence of citratonickelate(II), the counter-cations being 
a mixture of Ni(H,O)z+ and hydrated Mg2' and Ca2+. 
Where the extracts contain only low concentrations of 
magnesium and calcium (< 1 % of the solid), as in the 
case of the latex of S. acziminata, the nickel analysis and 
electrophoresis indicate that Ni(H,O);' is the major 
cationic constituent. 
The Ni(H,O)z+ can be removed by passing the 
extracts through a weak cation exchange gel (Sephadex 
CM-25). The absorption spectra (340-740 nm) of. the 
resulting solutions correspond to those of a 2:  1 citrate/ 
nickel solution and differ from those of Ni(H20)zf and 
1 : 1 citrate/nickel solutions. 
We have observed that the nickel electrophoretograms 
of Tiffin [2] closely resemble those of plant extracts 
shown in Fig. 1. It appears probable that the nickel 
carrier in the plants studied by Tiffin is also a citrato- 
complex, and that translocation of nickel by citrate is 
not confined to those plants which accumulate high 
concentrations, but is a more universal occurrence. 
In attempting to elucidate the structure of a metal- 
organic complex in plant material the question always 
arises as to whether the complex was formed metabolically 
or whether it is an artefact of the extraction process. One 
way to approach this problem is to examine the spatial 
distribution of metal concentrations and then check to  
see whether the organic ligand has the same distribution. 
Electron-probe measurements on leaves of several of the 
New Caledonian accumulator species showed that 
nickel was always distributed in a uniform manner 
without concentration at specific sites and the above 
method of approach was clearly inoperable. However, 
since the nickel-citrate complex was clearly evident in the 
latex of S. acuminata which was used directly without use 
of extraction procedures, there is no reason to suppose 
that presence of the same complex in the leaves of this 
and other species is the result of non-metabolic formation. 
If, as has been suggested, translocation of nickel by 
citrate is a universal characteristic of most plant species, 
this finding should have significance in fields other than 
plant chemistry or plant physiology. Considerable re- 
search is now under way throughout the world on the 
problem of extracting nickel from ores by low-energy 
processes, and it may be possible that complexing of this 
metal as citrate is a mechanism which should be explored 
for such a purpose. 
EXPERIMENTAL 
Extraction of complexes. To prevent Ni complexes appearing 
as an artefact in the separation process extraction of macerated 
fresh or freeze-dried leaf material was carried out with HzO at 
room temp. 50-70% of the total Ni could readily be extracted 
under these conditions. The extracts were shaken with CHC1,- 
n-BuOH (1O:l) to remove high molecular weight compounds of 
low polarity. Negligible amounts of Ni were lost in this process. 
Chromatography. The aq. soh was reduced in vol. and passed 
through a Sephadex G-10 or G-25 column. The latex from S .  
acuminata was passed through a filter paper directly on to a 
Sephadex G-50 column. H,O was used to elute the columns, 
again to reduce the possibility of complex formation between 
the extracted Ni and any added reagents. The green Ni-contain- 
ing fraction was recycled through the column several times 
before being taken to dryness at room temp. 
IR spectra. Because microanalysis of the solids showed the N 
content to be immeasurably small, the possibility of amino acids 
being involved was eliminated and IR spectra were recorded to 
identify frequency characteristics of specific groupings. 
GLC-MS. In the presence of trifluoroacetic acid, 10-30 mg 
of the Ni complex was reacted with N-(trimethylsily1)imidazole 
in dimethyl sulphoxide as solvent, giving a trimethylsilyl (TMS) 
derivative of the organic ligand in the upper clear layer of the 
reaction mixture. GLC (1709 glass column with 1.5 % OV-101 
on Chromosorb W, AW-DMCS, 100-120 mesh) using a 1 p1 
aliquot of the upper layer, was used to check the TMS derivatives. 
The gas chromatograph was connected to a MS which was run 
at 20 eV, separator temp. 160". 
High-voltage electrophoresis. High voltage paper electro- 
phoresis was carried out on the extracted Ni complexes, and 
on Ni chloride and Ni citrate solutions, using a Savant-type 
apparatus. Experimental conditions are given in the caption 
to Fig. 1. 
Identification of a Citrato-Complex from Nickel-Accumulating Plants 1505 
Acknowledgements-The authors are indebted to Drs. L. Nixon, 
I. G. Andrew, R. T. Gallagher, and Professor R. Hodges for 
advice and assistance. 
REFERENCES 
1. Tiffin, L. O. (1972) in Micronutrients in Agriculture (Mort- 
vedt, J. J., Giordano, P. M. and Lindsay, W. L. eds.), p. 199. 
Soil Sci. Soc. Amer., Madison, Wis. 
2. Tiffin, L. O. (1971) Plant Physiol. 48, 273. 
3. Bremner, 1. and Knight, A. H. (1970) Brit. J .  Nutr. 24, 279. 
4. Van Goor, B. J. and Wiersma, D. (1976) Physiol. Plailtaruin 
5. Tiffin, L. O. (1970) Plant Physiol. 45, 280. 
6. Thompson, J. F. and Tiffin, L. O. (1974) Plant Physiol. 
7. Lyon, G. L., Brooks, R. R. and Peterson, P. J. (1969) Planta 
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Suppl. 23. 
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8. Farago, M. E., Clark, A. J. and Pitt, M. J. (1975) Co-ord. 
9. Kelly, P. C., Brooks, R. R., Dilli, S. and Jaffré, T. (1975) 
10. Severne, B. C. (1972) PhD Thesis, Massey University, 
11. Pelosi, P., Galoppini, C. and Vergnano Gambi, O. (1974) 
12. Pelosi, P., Fiorentini, R. and Galoppini, C. (1976) Agric. 
13. Jaffré T., Brooks, R. R., Lee, J. and Reeves, R. D. (1976) 
14. Brooks, R. R., Lee, J., Reeves, R. D. and Jaffré, T. (1977) 
15. Petersson, G. (1970) Tetrahedron 26, 3413. 
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I 


Isolation and identification of a citratocomplex of nickel from nickel-accumulating plants

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