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III. Physiological Role of Molybdenum in Plants
MOLYBDENUM IN SOILS, PLANTS, A N D ANIMALS
tion of NO,- and in the fixation of N,, respectively. This section will include a
discussion of these two enzymes (molybdoproteins) as they function in the plant
The first molybdoprotein, nitrate reductase, is known to require Mo and flavin
for its activity and in the reduction of NO,- to No,- as follows:
Reduced N A D
+ NO,- + H,O
where NAD is nicotinamide adenine dinucleotide. The reduction mechanism
from NO,- to NOz- has been proposed by Nicholas (1975) as
+ 2H' + 2 e - + NO,- + H,O
+ H+ + 2e- -+ NO,- + OH-
Reaction (2) is based on the acidic half reaction, whereas reaction (3) allows for
OH- participation at physiological pH.
Nitrate reductase is found in most plant species as well as fungi and bacteria
(Price et al., 1972). The increased Mo requirement of most plants grown on
No,--N compared with NH,+-N can be almost completely accounted for by the
Mo in nitrate reductase (Evans, 1956).
The other major known molybdoprotein of plants, nitrogenase, fixes elemental
nitrogen in the form of NH,, which is then assimilated by the plant (Koch ef al.,
1967). The role of Mo in the fixation of N, has been reviewed in detail by Chatt
(1974). The unique role of Mo in biological systems is exemplified by nitrogenase, the enzyme that converts N, into NH, at room temperature and normal
pressure (Schrauzer, 1976). The nitrogenase is an enzyme complex composed of
two distinct components that combine to reduce N, to NH, [reaction (4)] or
acetylene to ethylene [reaction (5)] (Nicholas, 1975):
+ 6H+ + 6 e C,H, + 2H+ + 2eN,
Nitrogenases have been isolated from a variety of different sources, for example, from Azotobacter vinelandii, Rhizobium japonicum, Azotobacter chroococcum, and Klebsiella pneumonianum (Schrauzer, 1976).
Recent studies by Agarwala et al. (1978) have shown that in addition to
reduced nitrate reductase activity, Mo deficiency in corn resulted in significantly
lower activities of catalase, aldolase, and alanine aminotransferase and higher
activities of peroxidase, P-glycerophosphatase, and ribonuclease.
In addition to the involvement of Mo in the fixation of N, and nitrate reduction, Mo is associated with other processes in plants. However, many of these
processes are interrelated with the two main functions. For example, experiments
of Malonosova (1 968) showed that addition of Mo to the soil resulted in better
development of lupine (Lupinus spp.) and increased weight of its roots and
nodules although the N, was not fixed. In this review of Russian work it was
UMESH C . GUFTA AND JOHN LIPSETT
reported that on soils containing little Mo, plants will develop many nodules on
roots, but N, is not fixed.
Merkel et al. (1975) showed that Mo deficiency in tomatoes decreased organic
nitrogen content of the leaves to the same degree that it decreased the organic
anion content of the leaves. This change was mainly in the contents of malate and
citrate. Studies by Anderson and Spencer (1950) showed that deficiency of Mo
decreased both the protein nitrogen and the nonprotein nitrogen percentages in
the clover. The percentage crude protein in a number of plant species has been
found to increase with optimum rates of Mo applied to the soil (Reddy, 1964).
Hagstrom and Berger (1965) found that applications of Mo increased the nodulation and nitrogen content of peas and soybeans. The effect of Mo in plants is to
increase the content of proteins and to create favorable conditions for the biosynthesis of nucleic acids (Peyve, 1969).
Inden (1975) stated that deficiency of Mo can cause plant chlorosis, which is
due to the inability of the plant to form chlorophyll. Further, since the deficiency
of Mo reduces the rate of NO3- reduction in plants, photosynthesis decreases
because the end products are not removed by combination with nitrogenous
compounds. Das Gupta and Basuchaudhuri (1977) found that Mo applications
enhanced the nitrate reductase activity in the rice (Oryza sativa L.) plant, particularly under the high-nitrogen nutrition. This ultimately led to greater concentration of reduced nitrogen and thereby created a concentration gradient for the
uptake and subsequently greater assimilation of nitrogen in the tissues as
suggested by Wardlaw (1968). This suggests that there exists a close functional
relationship between the nitrate reductase activity and chlorophyll content as
observed by Das Gupta and Basuchaudhuri (1977). The chlorophyll content of
corn has been found to decrease due to a deficiency of Mo (Agarwala et al.,
1978). Molybdenum was also considered to be associated with the metabolism of
Fe and phosphoric acid (Inden, 1975).
Premature sprouting of maize grain on the cob was shown to be controlled in
glasshouse and field experiments by the Mo concentration of grain (Tanner,
1978). It was found that when the Mo concentration of grain fell below 0.05
ppm, sprouting occurred, the severity of which was enhanced by heavy, late
sidedress of nitrogenous fertilizer. The explanation offered was that Mo-deficient
plants have a decreased ability to reduce NO3- and consequent accumulation of
inorganic NO3- promotes sprouting.
Under Mo deficiency, plants accumulate low-molecular-weight nitrogen compounds and may have defective vascular bundles in the midrib in young leaves.
There is a deposition of a brown substance on tissue surface and in intercellular
spaces and cells (Busslar, 1970).
Hewitt (195 1 ) pointed out that a low concentration of ascorbic acid in tissues is
a characteristic of Mo deficiency in a number of species. Agarwala (1952) also
demonstrated that cauliflower plants grown with various nitrogen sources, in-
MOLYBDENUM IN SOILS, PLANTS, AND ANIMALS
cluding urea and ammonium sulfate, developed characteristic Mo deficiency
symptoms known as “whiptail” and contained reduced concentrations of ascorbic acid. Subsequent studies by Agarwala and Hewitt (1955) showed that Mo
deficiency decreased the total and reducing sugars in the leaves of young cauliflower plants.
In nutrient culture studies in flax (Linurn usitatissirnurn L.), Mo has been
found to be closely associated with the regulation of the deleterious effect of Mn,
Zn, Cu, Ni, or Co on the physiological availability of Fe to the plant (Millikan,
IV. DETERMINATION OF MOLYBDENUM IN SOILS
Thiocyanate and dithiol are the two most commonly used reagents employed
in the colorimetric determination of Mo from biological materials. The dithiol
method used by Piper and Beckwith (1948), Clark and Axley (1955), and
Bingley (1963) for plants and soils has been found to be more sensitive and
precise than the thiocyanate method because the green-colored complex formed
between Mo and dithiol was stable for at least 24 hours (Gupta and MacKay,
1965a). Later, Fuge (1970) developed a rapid and simple method in which a
Technicon AutoAnalyzer is used for the determination of Mo on the basis of its
catalytic action on the potassium iodide-hydrogen peroxide reaction. Fernandez
et al. (1978) used 2,2-dihydroxybenzophenone reagent and considered it to be
superior to the most commonly used Mo-thiocyanate complex method because
an extra step is not necessary and the results are more reproducible. Molybdenum
has also been determined spectrochemically after chemical concentration, using
the cathode-layer arc technique (Mitchell, 1974) and polarography (Dekhkankhodzhayeva and Kruglova, 1972). Trace quantities of Mo have been determined
by atomic absorption spectroscopy (AAS), both flameless (Henning and Jackson,
1973; Jarrel and Dawson, 1978) and flame (Khan et al., 1979). Little and
Kemdge (1978) used a carbon rod analyzer for determining very low levels of
Mo. The high temperature required to atomize Mo in this procedure makes it
easy to remove matrix materials during the ashing phase.
The colorimetric method using dithiol and the most recently used AAS are
probably the most commonly used techniques for determining Mo in soil and
plant materials. The detection limits for determination of Mo by AAS using
flame and graphite furnace have been found to be 10 and 2 ng/ml, respectively
(Khan et al., 1979). Using the dithiol colorimetric method (Gupta and MacKay,
1965a), the satisfactory detection limit is about 20 nglml. The recovery of Mo
added to the plant material as determined by these two methods has been found to
range from 92 to 95%.
UMESH C. GUPTA AND JOHN LIPSETT
I N SOILS
The most common method for extracting Mo from soils is by perchioric acid
digestion (Reisenauer, 1965). Dry ashing of soil and the extraction of ash using
concentrated acids was employed for determining total Mo in soils by Perrin
(1946) and Grigg (1953a). Total Mo has also been extracted by Na&O, fusion of
soil (Purvis and Peterson, 1956). Unpublished data of the first author of this
article showed that such extracts contained large quantities of interfering materials and required purification, which is time consuming. Little and Kerridge
(1978) used HF-HC104 digestion for determining total Mo in soils.
As for other plant nutrients, total Mo content of soils, except for very low
levels, is generally not a good indicator of plant Mo availability (Little and
Kemdge, 1978; Williams, 1971). Available Mo content has not been found to
be closely related to the total Mo content of soils (Stone and Jencks, 1963).
However, soil with a total Mo content of more than 20 ppm may be regarded as
potentially “teart” (producing Cu deficiency in animals) in Scotland (Williams,
1971). Soils with low total Mo and neutral to alkaline pH may be depleted by
many years of intensive cropping (Davies, 1956). Liming can correct Mo deficiency; therefore an estimate of total Mo content may provide some indication of
the Mo supplying power of acid soils. Details of the effect of liming on Mo
availability will be dealt with in Section VI,B.
Little information exists on the levels of Mo in various soils but, in general,
contents of 0.5-5 ppm are normal (Robinson and Alexander, 1953; Williams,
1971) and in agreement with the relative abundance of Mo in the lithosphere (2.3
ppm), whereas figures of 0.5 pprn or less would be considered particularly low
(Williams, 1971). The Mo content of a few soils selected from areas of Canada
close to industrial plants ranged from 1 .O to 11.3 ppm (Warren, 1973). MacLean
and Langille (1973) reported that the Mo content of Nova Scotia (Canada) podzol
soils ranged from 0.05 to 12.1 ppm.
The presence of extremely small quantities of Mo in the soil, the influence of
chemical characteristics of soils (Karimian and Cox, 1979), the importance of
seed reserves (Gurley and Giddens, 1969), and the possibility that seed reserves
may mask a deficiency in the soil make the problem of determining Mo
availability more difficult than for the other micronutrients. The first report on
the available Mo in soils, which related extracted Mo to plant uptake, was by
Grigg (1953b) in New Zealand. This involved an acid oxalate extractant buffered
at pH 3.3. The responses and lack of responses as related to Mo extracted by
Grigg’s reagent for a number of crops have been summarized by Reisenauer