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12 Methods to improve the crop-delivery of
minerals to humans and livestock
Michael A. Grusak and Ismail Cakmak
12.1 Introduction
Humans and other animals are dependent on plant species to provide them with
dietary minerals. Plants can contain a broad range of mineral elements, but
concentrations in any one plant will vary depending on species, genotype and
environmental constraints. In theory, a balanced diet containing several plant
food sources will provide an adequate dietary intake of all essential minerals
for any given species (Dwyer, 1991; American Dietetic Association, 2002). In
practice, however, diets are not always diverse enough, or consumed in sufficient
quantities, to assure adequate intake of all minerals. This situation is especially
prevalent in low-income populations of the human species throughout the developing world, where total caloric intake is low, and diets are restricted to one
or two staple foods that are often a poor source of several minerals (Food and
Agriculture Organization of the United Nations, 2000; Underwood, 2000). Similarly, for livestock, mineral needs for optimal growth or productivity are rarely
met by plant foods alone; supplemental minerals often are added to animal feeds
(Pond et al., 1995).
To help provide higher quantities of plant-based dietary minerals, researchers
have been working to enhance the mineral density of plant foods. This is not
proving to be an easy task, as minerals must be acquired from the rhizosphere,
and are partitioned to edible tissues via a complex, integrated series of shortand long-distance transport events (Grusak, 2002a). While some gains have been
realized through conventional breeding, especially for micronutrients, continued
efforts to understand the molecular mechanisms and regulation of plant mineral
nutrition are essential if we wish to make significant improvements in the food
supply. Thankfully, genomic studies are beginning to provide us with some of the
necessary knowledge and tools to effect these changes. In this chapter, we will
discuss the importance of plants in the dietary food chain, why plant mineral
research is important and must be expanded, and how our existing genomic
(and conventional) technologies can be applied to initiate improvements in plant
mineral content. We have attempted to present these issues within a conceptual
framework to discuss how nutritional genomics, per se, can facilitate plant
improvement. Several other articles on the role of agriculture and molecular
genetics to increase plant mineral content are available (Grusak & DellaPenna,
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1999; Frossard et al., 2000; Cakmak, 2002; Cakmak et al., 2002; Williams,
2003; Poletti et al., 2004; Welch & Graham, 2004).
12.2 Plants as sources of dietary minerals
12.2.1 Mineral nutrition for humans
To understand better how plant mineral content might be improved, it is first
worth noting what humans require and what plant foods can provide. There are
16 mineral elements deemed essential for humans. These include the macronutrients N, S, K, Ca, P, Cl, Na and Mg, along with the micronutrients Fe, Zn,
Mn, Cu, Mo, Cr, I and Se (National Research Council [US] Food and Nutrition
Board, 1989) (see Table 12.1). Dietary proteins, peptides and free amino acids
are the predominant source of N and S, and in fact, much of the required N and
S must be obtained as essential amino acids (Reeds & Beckett, 1996). The other
macro- and micronutrients are obtained and absorbed in various organic and
inorganic forms, including free ions. Additionally, elements such as B, Ni, Si
and V have been suggested as human-essential, but evidence for these remains
circumstantial (Nielsen, 1996). Nonetheless, their occurrence in plant foods,
Table 12.1 Human mineral requirements and examples of mineral content in plant food sources (per
100 g f. wt food)
Required Mineral
Maximum Adult
US RDA1
Content in cooked
white rice2
Content in cooked
black bean2
Content in raw
spinach2
Potassium (K)
Calcium (Ca)
Phosphorus (P)
Chloride (Cl)
Sodium (Na)
Magnesium (Mg)
Iron (Fe)
Zinc (Zn)
Manganese (Mn)
Copper (Cu)
Molybdenum (Mo)
Chromium (Cr)
Iodine (I)
Selenium (Se)
2000 mg
1200 mg
1200 mg
750 mg
500 mg
350 mg
15 mg
15 mg
2–5 mg
1.5–3 mg
75–250 g
50–200 g
150 g
70 g
10 mg
2 mg
8 mg
NA3
5 mg
5 mg
0.14 mg
0.41 mg
0.26 mg
0.05 mg
NA
NA
NA
5.6 g
355 mg
27 mg
140 mg
NA
237 mg
70 mg
2.10 mg
1.12 mg
0.44 mg
0.21 mg
NA
NA
NA
1.2 g
558 mg
99 mg
49 mg
NA
79 mg
79 mg
2.71 mg
0.53 mg
0.90 mg
0.13 mg
NA
NA
NA
1.0 g
1 Recommended dietary allowances (RDA) are the daily levels of intake of essential nutrients judged to
be adequate to meet the known nutrient needs of practically all healthy persons. Values presented are the
highest RDA either for male or female adults, excluding pregnant or lactating women. Values are from
National Research Council (US) Food and Nutrition Board (1989).
2 Values are from US Department of Agriculture, Agricultural Research Service (2001).
3 NA: not available.
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along with health-beneficial elements like fluoride (for the prevention of dental
caries), has dietary significance.
Plant foods can provide all of these elements, especially those that have been
determined as essential for the growth and reproductive development of plants
themselves. The plant-essential elements are N, S, P, K, Ca, Mg, Cl, Fe, Zn,
Mn, Cu, B, Mo and Ni (Eskew et al., 1983; Marschner, 1995). Because these
14 are required, all plants acquire them from soils through various transport
mechanisms, and they should be found in all plant foods. Elements not identified
as plant essential, but which are essential for humans (e.g. Na, Cr, I, Se, Si and
V), can enter the plant through various non-selective transport mechanisms when
these minerals are available in the soil (Kochian, 1991). Fortunately, for humans
and livestock, these other minerals often do make their way into the food supply
(Kabata-Pendias & Pendias, 1992). However, for any of these elements (plant
essential or non-essential), their content in plant foods can be quite variable
(Table 12.1). Mineral concentrations can differ across tissues within a single
plant, across genotypes of a given species, or more broadly across species.
Thus, although plants have the potential to deliver many dietary minerals, that
delivery is not always optimal in any given food source.
12.2.2 Recommended intake versus actual intake in humans
Many governments provide dietary recommendations for the daily intake of minerals, vitamins, protein, lipids, carbohydrates, water and other health-beneficial
compounds (Harper, 1985, 1987). These recommendations vary across the life
span, as well as with different physiological states. Infants and children have
lower requirements for most minerals, relative to adults, and pregnant or lactating women have higher needs for some minerals relative to men of the same age
(National Research Council [US] Food and Nutrition Board, 1989). Infectious
diseases can also promote different mineral requirements, especially in the case
of intestinal parasites where nutrient absorption is impaired (Thurnham, 1997;
Hoste, 2001). Unfortunately, not all individuals achieve these recommended intakes, either because of personal choice or because of environmental constraints
(Anonymous, 1998; Edmonds et al., 2001). For instance, in developed countries, most mineral intake inadequacies exist not because of a lack of food, but
rather because of behavior-influenced decisions in food selection (Baranowski
et al., 1999). Total caloric intake often exceeds recommended guidelines, as
evidenced by the rising global incidence of obesity (Rössner, 2002); unfortunately, the prevalence of and preference for processed foods, often with low
mineral density, does not provide adequate quantities of all minerals. In the
developing world, large segments of the global population have food intakes
that are severely low, leading to malnutrition of energy, protein, minerals, or
other micronutrients (Food and Agriculture Organization of the United Nations,
2000). Poverty, in conjunction with the high cost of vegetables and animal food
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products, leads many to subsist on a diet predominated by staple foods (e.g. rice,
wheat, maize, bean, cassava and sweet potato) that have low concentrations of
many minerals.
Because of these low intakes of food and/or minerals, several food-related
nutrient-deficiency diseases can be found throughout the world. Iron deficiency,
which is believed to affect 30–40% of the world’s population, has a deleterious
effect on cognitive development in children and on work productivity in adults
(Yip & Dallman, 1996). Zinc deficiency, also believed to occur throughout a
large percentage of the world’s population (but firm estimates are unavailable
owing to the lack of good biomarkers), causes stunting in infancy, delayed maturity of reproductive development and reproductive organs, impairments in brain
development and function, and increases susceptibility to various pathogenic
diseases (Hambidge, 2000; Sandstead, 2003). Iodine deficiency is another nutritional disease, with an estimated global incidence of 13%, but with 38% of the
world’s population currently at risk for iodine deficiency disorders (World Health
Organization, 1999). Low I status can lead to irreversible mental retardation in
children and goiters in adults (Stanbury, 1996). Besides these micronutrients, a
deficiency in Ca can be found in certain regions of the world. The incidence of nutritional rickets, a disease in which insufficient Ca intake leads to stunting and severe bone deformities, is quite high in parts of India and Africa (Thacher, 2003);
it also is starting to be observed in developed countries (DeLucia et al., 2003).
Finally, Se deficiencies have been seen in parts of China (Fordyce et al., 2000),
and are an increasing concern in several European countries (Arthur, 2003).
12.2.3 Bioavailability
Although total mineral intake is an important determinant of mineral adequacy
(in humans and livestock), not all ingested minerals are completely absorbed
and utilized. Bioavailability is a term used to describe the digestion, absorption
and subsequent utilization of dietary compounds (Linder, 1991). It encompasses
all the physical, chemical and enzymatic processes that contribute to the breakdown of food, and includes the secretory processes and membrane transporters
in the gut that facilitate nutrient absorption and trafficking into the body. For
minerals within a given food source, their bioavailability depends on the types
and quantities of promotive and inhibitory compounds that are found within that
food, or that are ingested concurrently from other foods in the meal. Calcium
bioavailability, for instance, can be as high as 30% from some foods, or as low
as 5% in foods where this mineral is found as Ca-oxalate crystals (Weaver &
Heaney, 1991). Dietary phytic acid (phytate) can influence bioavailability of
several minerals (e.g. Fe, Zn, Ca), because of its capacity to form insoluble
precipitates (Wise, 1995). As with Ca-oxalate crystals, these insoluble components are unavailable to membrane transporters on the surface of enterocytes
(absorptive cells of the intestinal epidermis), as food moves through the gut.
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Several organic molecules can also influence mineral bioavailability. These
include tannins and various polyphenolics as inhibitors, or ascorbic acid and
S-amino acids as promoters (Welch & Graham, 2004). Although efforts have
been undertaken to manipulate these compounds in plants, especially the reduction of tannins, the potential also exists to manipulate inorganic constituents
directly. A better understanding of how Ca is channeled to and/or sequestered
in Ca-oxalate crystals could allow us to reduce levels of this mineral salt in
plants (Nakata, 2003), thereby improving Ca absorption. Similarly, a sound
knowledge base in P nutrition and phytate metabolism will allow us to lower
phytate levels in target tissues, such as has been achieved with low-phytate
maize mutants (Raboy, 2001), or has been attempted with the engineering of the
phytate-degrading enzyme, phytase (Lucca et al., 2001; Brinch-Pedersen et al.,
2003). Clearly, continued research in specific areas of plant mineral nutrition is
needed to effect these types of changes.
12.2.4 Mineral nutrition for livestock
The essential minerals required by livestock are the same as those needed by
humans (Pond et al., 1995). However, the daily requirements for specific minerals are quite different. Not only do these vary considerably from one animal
species to the next, but the basis for establishing mineral needs is also somewhat different in livestock than in humans. For people, the nutritional goal is
to provide sufficient minerals that will minimize deficiencies in the population
and will maintain adequate growth in infants and children, or stable body mass
and composition in adults. For livestock, although mineral intakes to prevent
deficiency have been studied in the past, current efforts are more focused on
the economics of production. Mineral intake levels often are set to maximize
growth (e.g. swine, beef cattle, broiler chickens) or to optimize productivity (e.g.
dairy cattle, laying hens) (Coleman & Moore, 2003). Thus, with recommended
mineral intakes high for certain livestock, it is common for commercial feed to
contain supplemental minerals (Pond et al., 1995). Efforts to enhance mineral
concentrations in plant foods would reduce the need for some of these additions,
and thereby lower costs to the livestock producer. Interestingly, these mineral
needs are not static, but are rather a moving target. The production potential
and nutritional requirements of animals are also under constant manipulation
through conventional breeding and biotechnology (Bonneau & Lavard, 1999).
The type of plant material fed to livestock differs, in part, from that consumed
by humans. Although cereals and grain legumes are fed widely to many animals,
forages are a significant part of some diets, especially for ruminant animals (Pond
et al., 1995; Reddy et al., 2003). Both monocots (e.g. Lolium spp., Pennisetum
spp.) and dicots (e.g. Medicago sativa, Trifolium spp.) serve as forage, and
thus plant scientists should not overlook these species as targets for mineral
improvement. In the case of forages, strategies to increase mineral concentration
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would be worthwhile, but efforts to ensure an appropriate balance of minerals
would be equally important. Grass tetany, for instance, is a serious and lifethreatening disease caused by an imbalance in plasma Mg that arises when
K/Mg ratios are elevated in forage grasses (Robinson et al., 1989; Dua & Care,
1995).
12.3 Conceptual strategies for mineral improvement
Plant nutritional science is at an exciting juncture because of the wealth of sequence information available for mineral-related genes in various model and
agronomic species, and because of the breadth of technologies available to
study the expression and function of relevant genes and gene products (Grusak,
2002b). Scientists have now identified membrane transporters for several mineral ions (Williams et al., 2000; White & Broadley, 2003), and these molecular
tools could aid efforts to enhance mineral density in plant foods, either through
genetic engineering or through marker-assisted selection (see below). However,
although knowledge of specific genes can be useful, it is important to remember
that any one protein product functions within a broader whole-plant system, and
can only move a mineral from point A to point B, or perhaps can only act to
sequester it within one compartment. No protein allows a plant to ‘make’ a mineral, unlike the situation with biosynthetic enzymes that can convert precursors
into other molecules of interest. Instead, all minerals must be acquired from
the soil matrix, must be moved to the xylem for delivery to transpiring shoot
tissues, must be transported across membranes for utilization by cells along the
root–shoot pathway, and in most cases must ultimately be partitioned to the
phloem pathway for long-distance transport to seeds.
Strategies for mineral improvement, therefore, must take into account the role
of several short- and long-distance transport processes, as well as the availability
of a given mineral in different tissues. One can think of the soil–plant continuum
as a series of compartments, in which different transporters provide specific
transfer capabilities for each mineral from one compartment to the next, and
each compartment has a unique storage capacity (i.e. pool size) for each mineral.
Total flux of a mineral from the soil to a terminal tissue in the plant (e.g. leaves
or seeds) will be determined both by the rate limitations of transport steps along
the pathway and by the size of the mineral pool in each successive compartment.
Pool size is an important issue, because it helps identify where increased
compartmental flux is needed within the whole-plant system. For instance, Fig.
12.1 shows a distribution of the partitioning of several minerals between shoot
vegetative and reproductive tissues in a mature pea plant. It can be seen that the
partitioning of some minerals to seeds is quite high. This means that efforts to
double the Fe or Cu concentration in pea seeds would require enhanced transport
into the plant to increase the vegetative pool of these minerals, and may require
elevated rates of transport from leaves to seeds (i.e. via phloem loading). On the
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CROP-DELIVERY OF MINERALS TO HUMANS AND LIVESTOCK
Seed Fraction
Vegetative Fraction
Fe
Zn
Cu
Mn
K
Mg
0
20
40
60
80
100
Mineral partitioning
Figure 12.1 Partitioning of total shoot minerals between seed and vegetative fractions (stems, leaves,
stipules, and pod walls) in a pea plant at harvest maturity. Data for cultivar Sparkle are presented as the
percentage of minerals measured in total shoot tissues (M.A. Grusak, unpublished observations).
other hand, a doubling of Zn, Mn, K, or Mg concentration in seeds theoretically
could be accomplished solely by increasing flux from leaves to seeds. Note that
phloem loading and transport are mentioned here, because seeds are surrounded
by tissues that block transpiration from the seed surface (e.g. all legumes, maize),
and thus there are no minerals imported via the xylem pathway (Grusak, 1994,
2002a).
For most minerals, further research is needed to identify and characterize the full inventory of molecular- and tissue-level components that contribute to whole-plant mineral dynamics. Thankfully, nutritional genomic studies are adding to this knowledge, especially in the areas of gene discovery and
sequence analysis, gene expression and functional analysis of proteins. More
information is also needed on existing variation in mineral concentrations, both
in edible and non-edible tissues. Once a complete picture of the rate limitations in any plant–mineral situation is available, one then can design rational
approaches to increase mineral concentrations. These approaches can include
the use of existing genetic variation, conventional breeding and marker-assisted
selection and/or transgenic technologies.
12.4 Exploiting existing genetic variation
Significant genotypic differences have been reported for seed concentrations
of minerals, especially for micronutrients in staple food crops such as wheat,
rice, maize and bean (see also Chapter 10). Such genotypic variation can be
exploited for improving food crops with enhanced levels of minerals, and in fact
is essential for a successful breeding approach. Because of the global problem of
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human micronutrient deficiencies, as noted above, a Micronutrient Project was
initiated with several partners within the Consultative Group on International
Agricultural Research (CGIAR) and other academic and government scientists
(Bouis, 1996; Bouis et al., 2000), to screen a large number of plant genotypes for
seed or root concentrations of Fe and Zn. Extensive genetic variation for these
micronutrient minerals has been reported for wheat (Monasterio & Graham,
2000), rice (Gregorio et al., 2000), maize (Bänziger & Long, 2000), bean (Beebe
et al., 2000) and cassava (Chavez et al., 2000) (Table 12.2). This genetic variation
is presently being exploited in the breeding programs at different CGIAR centers
in the framework of the HarvestPlus Challenge Program coordinated by IFPRI
(International Food Policy Research Institute) and CIAT (International Center
for Tropical Agriculture) (Bouis, 2003). Below, several examples are given for
the extent of genotypic variation found for mineral nutrients, and the ways that
this variation can be used to enhance mineral content in plant foods.
12.4.1 Wheat
Throughout much of the world, wheat contributes greatly to energy, protein
and mineral intake, and in some regions it is the predominant staple food crop,
providing a major portion of daily calories (Fig. 12.2). Because of this extensive
consumption, efforts to improve wheat with enhanced levels of micronutrients
can play a paramount role in reducing global micronutrient deficiencies.
Data on genetic variation for micronutrients in wheat and related species is
available from screening studies realized at CIMMYT (International Maize and
Wheat Improvement Center). In a germplasm study with 505 genotypes including high-yielding bread wheat and durum wheat genotypes, triticale, synthetic
wheats and several other genetic materials grown in different locations, seed
concentrations of Fe and Zn ranged from 20 to 59 with an average of 34 mg Fe
kg−1 d. wt and from 16 to 67 with an average of 34 mg Zn kg−1 d. wt, respectively (Table 12.2). From this screening study, a subset of genotypes with very
low and very high micronutrient concentrations (170 genotypes) was chosen for
concurrent planting at the same location. The genetic variation for Zn and Fe in
this subset was very similar to the variation found with the 505 genotypes (Table
12.2). In further germplasm studies that included primitive and wild species of
wheat, the genetic variation was much larger, especially in the case of the primitive wheat, Triticum dicoccum (Monasterio & Graham, 2000). At CIMMYT,
breeders have recently initiated a program using promising genotypes of wild
relatives of wheat (Aegilops tauschii), primitive wheat (T. dicoccum) and prebreeding lines, to transfer alleles contributing to elevated seed concentrations of
Zn and Fe into high-yielding elite wheat cultivars.
In the case of wild wheats, natural variation for micronutrients is more extensive and thus these are more promising for a successful breeding program.
Wild wheats and wild relatives of wheat are widespread in different populations
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16
15
23
15
15
18
16
–
21
39
4
140
350
250
51
416
100
100
195
119
1031
20
20
64
9
43
54
22
25
27
58
42
58
40
50
67
65
92
Max
52
6
29
35
30
21
22
21
24
25
26
32
34
35
41
Mean
62
8
–
34
18
10
27
19
8
8
9
11
20
25
32
Min
155
13
96
89
59
17
57
39
24
17
24
23
59
56
73
Max
94
10
60
55
26
13
32
24
13
11
13
15
34
37
43
Mean
Iron (mg kg−1 d. wt)
Chavez et al., 2000
Chavez et al., 2000
Beebe et al., 2000
Beebe et al., 2000
Bänziger & Long, 2000
Bänziger & Long, 2000
Bänziger & Long, 2000
Bänziger & Long, 2000
Gregorio et al., 2000
Gregorio et al., 2000
Gregorio et al., 2000
Gregorio et al., 2000
Monasterio & Graham, 2000
Monasterio & Graham, 2000
Monasterio & Graham, 2000
Reference
subset of mixed genotypes selected after initial screening of the 505 genotypes grown at different locations (see Monasterio & Graham, 2000).
16
25
26
505
170
154
Min
Zinc (mg kg−1 d. wt)
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1 This
Wheat grown at CIMMYT
Mixed genotypes
Selected genotypes1
Pre-breeding lines
Rice genotypes grown at IRRI
Traditional and improved
IR breeding
Tropical japonicas
Aromatic rice
Maize grown at CIMMYT
Landraces
Germplasm pools
Breeding germplasms
Breeding germplasms
Bean grown at CIAT
Wild genotypes
Cultivated genotypes
Cassava grown at CIAT
Leaves
Roots
Plant species
Number of
genotypes
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Table 12.2 Ranges in seed concentrations of zinc and iron in various state food crops grown in field of different Consultative Group on International
Agricultural Research (CGIAR) centers
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70%
60%
50%
40%
30%
20%
10%
D
Tu Taj
rk ikis
m
K en tan
yr i
gh sta
A yzs n
ze ta
A rb n
fg a
ha ija
ni n
s
A tan
lg
A eri
rm a
e
Tu nia
ni
si
a
K
az Sy
ak ria
U h
zb st
ek an
M ista
or n
oc
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Tu n
r
G key
eo
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ev
e
D lop
EU
ev in
el g
op C US
ed ou A
C ntr
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nt s
ri
W es
or
ld
0%
Figure 12.2 Daily caloric intake from wheat in different countries and regions. Source: Food and Agriculture Organization Database 2003; compiled by H.J. Braun, International Maize and Wheat Improvement
Center (CIMMYT), Turkey.
throughout the Fertile Crescent Region (e.g. Iraq, Turkey, Syria, Lebanon, Israel
and Jordan), an area where Zn deficiency is a potential problem in soils (Cakmak
et al., 1999; Hacisalihoglu & Kochian, 2003). Many of the wild wheats have
originated from Turkey, where Zn-deficient soils are common, and it is likely
that wild wheats from Turkey have a high genetic capacity for the acquisition
of soil Zn. Of the wild wheat species studied, Triticum dicoccoides, a tetraploid
wheat, showed substantial variation for micronutrients, and especially for Zn
(Cakmak et al., 2000). In different T. dicoccoides accessions collected from
the Fertile Crescent region (nearly 800 accessions coming mainly from Israel
and Turkey), Fe and Zn concentrations of seeds varied from 15 to 96 mg Fe
kg−1 d. wt and from 30 to 118 mg Zn kg−1 d. wt, respectively (Cakmak et al.,
in press)). An analysis of 110 T. dicoccoides accessions subsequently grown
in a greenhouse showed genotypic variation for Zn that was particularly high
(14–190 mg Zn kg−1 d. wt); a similar range of variation was not found for other
mineral nutrients. The accessions having the highest seed Zn concentrations
also had higher seed size or seed weight, resulting in the highest measured
total Zn contents per seed (up to 7 g per seed). These results indicate that
high Zn concentration in seeds is not a consequence of seed size (i.e. not a
concentration effect of small seeds). It seems very likely that T. dicoccoides
represents a very valuable genetic resource for improving Zn concentration of
cultivated wheat, and several breeding efforts are in progress to exploit this
species to enhance Zn concentrations in seeds of high-yielding wheat cultivars
in Turkey and Israel. This species also could be an important source of alleles for
metal-related genes, especially orthologs of Zn transporters in the ZIP family
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(Guerinot, 2000; Gross et al., 2003). In fact, sequence polymorphisms in these
genes (between T. aestivum and T. dicoccoides) could provide useful molecular
markers for continued breeding efforts.
12.4.2 Rice
Rice is a major staple food crop for nearly 3 billion people, and contributes
to protein and carbohydrate intake mainly in Asia, but also in parts of Africa
(Khush, 1997). Mineral concentrations in rice seeds are generally low, and
mineral levels are diminished even further after milling and polishing, which
removes the nutrient-rich aleurone layer and embryo. The remaining endosperm
contains low levels of most minerals, especially the micronutrient metals.
Efforts at the International Rice Research Institute (IRRI) have been conducted to screen a large number of rice germplasm for genetic variation in
micronutrient concentrations. A total of 1138 genotypes collected from different countries were analyzed for Zn and Fe concentration after growing them
under the same environmental conditions. The genetic variation ranged from
6 to 24 mg kg−1 d. wt, with a mean value of 12 mg kg−1 d. wt for Fe, and from
15 to 58 mg kg−1 d. wt, with a mean value of 25 mg kg−1 d. wt for Zn (Gregorio
et al., 2000). Among the rice genotypes tested, new breeding lines and traditional lines had the lowest, and aromatic rice genotypes had the highest levels
of micronutrients (Table 12.2). Aromatic rice genotypes contain consistently
more Fe and Zn than do the nonaromatic genotypes. Seven aromatic and seven
nonaromatic rice genotypes grown under the same conditions demonstrated
average Fe and Zn concentrations that were 18 and 32 mg kg−1 d. wt for aromatic and 11 and 21 mg kg−1 d. wt for nonaromatic rice genotypes, respectively
(Graham et al., 1997). These results point to aromatic rice germplasm as promising genetic resources for improving micronutrient levels in rice.
12.4.3 Maize
Maize is a major staple food crop for many people living in Africa and the Americas, and is an important feed for livestock (Byerlee & Heisey, 1996). Unfortunately, maize-based foods and feeds are very low in micronutrient concentrations
and rich in phytic acid, which limits the biological utilization of many minerals.
Information on genetic variability for mineral nutrients in maize seed is available
from screening studies realized at CIMMYT and at the International Institute
of Tropical Agriculture (IITA) (Bänziger & Long, 2000; Maziya-Dixon et al.,
2000; Oikeh et al., 2003). Over 1800 maize genotypes screened for seed micronutrients in field experiments established in Mexico and Zimbabwe showed
concentrations ranging from 10 to 63 mg kg−1 d. wt and from 13 to 58 mg kg−1
d. wt, for Fe and Zn, respectively (Bänziger & Long, 2000) (subsets of these data
are presented in Table 12.2). However, the maize genotypes demonstrating elevated levels of Fe and Zn were associated with very low grain yield. According
to Bänziger and Long (2000), environmental factors contributed greatly to the
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genetic variation found for Zn and Fe in maize. Recently, Oikeh et al. (2003) also
found significant variation for Fe and Zn concentrations in seeds of 49 maize
genotypes grown at different locations in Nigeria. The variation in kernel Fe and
Zn concentrations were between 17 and 24 mg kg−1 d. wt and between 17 and
25 mg kg−1 d. wt respectively, and were not influenced by location. The more
promising genotypes maintained high levels of seed Fe and Zn across different
environments.
12.4.4 Bean
Grain legumes, especially common bean, are an important food source for many
people living in Latin America and Africa and can provide varying levels of
minerals, protein and carbohydrates (Wang et al., 2003). In some regions, beans
and cereals are consumed together, but their combined intake still does not meet
daily requirements for several minerals. Thus, there is a clear need for genetic
improvement of beans with enhanced levels of minerals and/or with reduced
levels of antinutrients (i.e. tannins, phytate) (Beebe et al., 2000).
Several examples exist of promising genetic variability for minerals in different bean germplasm. Studies conducted at CIAT, using a common bean collection comprising 119 wild and 1031 cultivated genotypes, showed seed Fe
concentrations ranging from 34 to 92 mg kg−1 d. wt, with an average of 35 mg
kg−1 d. wt, and seed Zn concentrations ranging from 21 to 54 mg kg−1 d. wt, with
an average of 35 mg kg−1 d. wt (Graham et al., 1999; Beebe et al., 2000). Interestingly, wild genotypes in these studies were not superior to cultivated genotypes
regarding the concentrations and variability of micronutrients (Table 12.2). By
contrast, it has been argued that wild and weedy common bean accessions collected in Mexico could be a promising genetic resource for improvements in seed
micronutrient levels (Guzman-Maldonado et al., 2000). However, in this study,
only two cultivated bean genotypes were compared with 70 accessions of wild
and weedy bean genotypes. Relative to the results obtained at CIAT, the genetic
variability for Zn in the genotypes from Mexico was smaller (ranging from 24
to 38 mg kg−1 d. wt), but for Fe the variation was greater (from 71 to 180 mg Fe
kg−1 d. wt). According to Beebe et al. (2000), although environmental factors
affect seed mineral concentrations to some degree, the effects of the genetic
components on elevated micronutrient concentrations is sufficiently stable and
expressed across different environments, such that breeding efforts for mineral
levels in bean should be fruitful. Similarly, studies of Ca levels in bean pods
suggest that breeding will be more effective in elevating pod Ca concentration
than would soil fertilization be (Miglioranza et al., 1997; Quintana et al., 1999).
12.4.5 Other crops
Although much of our discussion has focused on micronutrient metals, this is
not meant to imply that genetic diversity for other minerals is lacking. In pea, an
important legume both for humans and livestock, seed mineral concentrations
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were found to vary both for micro- and macroelements. Nearly 500 accessions of
the Pisum Core Collection, a genetically diverse subset of the entire US Department of Agriculture, Agricultural Research Service (USDA/ARS) pea collection (Western Regional Plant Introduction Station, Pullman, Washington, USA),
were grown under uniform, nutrient-replete conditions in a greenhouse (M.A.
Grusak, unpublished observations, 2003). Concentrations of the microelements
Fe, Zn, Mn and Cu varied 4.5-fold, 6.6-fold, 6.8-fold and 10.1-fold, respectively,
while concentrations of the macroelements Ca, Mg, K and P varied 9.1-fold,
2.3-fold, 2.8-fold and 3.6-fold, respectively. Specific mineral data for individual accessions are available at the Germplasm Resources Information Network
(GRIN) Web site (http://www.ars-grin.gov/cgi-bin/npgs/html/desclist.pl?177).
Similarly, for spinach, a leafy vegetable grown throughout the world, genetic
diversity for leaf mineral concentrations was shown to be quite broad. Almost
the entire USDA/ARS spinach collection (North Central Regional Plant Introduction Station, Ames, Iowa, USA) was grown under uniform, nutrientreplete conditions in a growth chamber (M.A. Grusak, unpublished observations, 2003). For 327 accessions, concentrations of the microelements Fe, Zn,
Mn and Cu varied 2.7-fold, 12.3-fold, 14.3-fold and 4.9-fold, respectively, while
concentrations of the macroelements Ca, Mg, K and P varied 3.8-fold, 2.9-fold,
3.9-fold and 2.6-fold, respectively. Leaf mineral data for individual spinach
accessions are also available at the GRIN Web site (http://www.ars-grin.gov/cgibin/npgs/html/desclist.pl?219).
12.5 Integrating genomic technologies for mineral improvement
Clearly, there is significant genetic diversity for almost any mineral of interest
within existing germplasm collections and/or other unique genetic populations.
This diversity offers tremendous opportunities to utilize various genomic resources and technologies, in an effort to manipulate mineral levels in plants.
Fortunately, following the successes and advances that came out of the sequencing of the Arabidopsis genome (Arabidopsis Genome Initiative, 2000), most
major crop species (e.g. the legumes Medicago truncatula and Lotus japonicus) have had genome projects in operation for several years. While the various
projects are currently at different levels of advancement, most are providing
crop-specific sequence information (expressed sequence tags [ESTs] from various tissues; genomic sequence), bacterial artificial chromosomes (BACs) for
BAC-end sequencing to generate physical maps (and for use in genomic sequencing) and gene indices (tentative consensus [TCs] sequences) compiled
from the computational analysis of ESTs. In addition, several genome projects
are generating molecular markers for mapping and the construction of genetic
maps, are developing comparative maps between the crop of interest and related species (and also often with Arabidopsis), are providing cDNA-based or
oligonucleotide-based microarrays for global gene expression studies, or are
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providing mutants for functional studies (e.g. T-DNA lines, or lines identified through high-throughput screening for induced point mutations [targeting
induced local lesions in genomes; TILLING]; McCallum et al., 2000; Scholte
et al., 2002). In Fig. 12.3, we present a generalized flowchart that includes many
of these tools and resources, and in the remainder of this chapter we will discuss
how they can impact cultivar development.
12.5.1 The Path to Gene Discovery
As noted earlier, mineral accretion in an edible tissue is determined by several
transport and/or storage processes, and in many cases, we are still attempting
to determine the rate-limiting process or molecular player that most influences
final mineral levels. This is where genetic diversity can make a significant impact. When mineral diversity is assessed in populations that are well mapped
with molecular markers (e.g. RILs; near introgressed lines [NILs]), the mineral
data can be statistically analyzed to identify quantitative trait loci (QTLs) associated with elevated mineral concentrations. This approach has been used to
locate QTLs for total P and phytate levels in Arabidopsis seeds (Bentsink et al.,
2003), as well as seed Fe and Zn concentrations in bean (Beebe et al., 2000).
When QTLs are identified in species that also have extensive genomic sequence
information available (e.g. Arabidopsis, rice), fine mapping in the vicinity of the
QTL can allow one to fairly quickly identify putative genes that have relevance
to the mineral trait. How quickly this can be done will depend on the original
density of markers in the QTL region, and how much additional crossing (to
generate NILs) and fine mapping will be needed to localize a short candidate
region of the genome. Alternatively, if full genome sequence is limited, but
BACs are available that are anchored to a physical map, fine-mapping followed
by shot-gun sequencing of specific BACs neighboring the QTL can also lead to
candidate genes (Lévy et al., 2004).
Subsequent verification of the putative mineral-related genes can involve
bioinformatic tools and/or functional methodologies (Borevitz & Chory, 2004).
For example, Basic Local Alignment Search Tool (BLAST) queries of sequence
databases (Altschul & Lipman, 1990) will provide ‘hits’ to similar sequences
entered in GenBank, from the same or other species. Although one always must
be cautious of gene annotations in GenBank (gene assignments are not refereed), BLAST results can be used to sort through the candidates. For mineral
phenomena, hits to genes encoding membrane transporters, metal-binding peptides, or transcription factors, to name a few, might be worth further attention,
especially those for which prior molecular information pertinent to a given mineral’s nutritional physiology is available. Protein function can then be verified
through heterologous expression of candidate genes in yeast (e.g. for membrane transporters; Lopéz-Millán et al., 2004), or, if available, knockout mutants
could be screened for altered mineral physiology. For species in which mutant
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Genetic
diversity
QTL
analyses
Genomic
analyses
Gene
discovery
Bioinformatic
and functional
analyses
Allele
discovery
Trait-specific
molecular
markers
Genetic
engineering
Improved
cultivar
Figure 12.3 Flowchart depicting the contribution of various nutritional genomics tools and resources that
can contribute to cultivar development.
populations have not been developed, one should consider using mutants in another plant system, if lines are available in a putative orthologous gene (Perry
et al., 2003). The tremendous strength of our nutritional genomic resources is that
we can and should move between different eukaryotic and prokaryotic systems
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to identify the molecular players in any mineral process (DellaPenna, 1999;
Delseny, 2004).
QTL analysis is not the only route to novel gene discovery. Microarrays can
provide comparisons of global gene expression in different tissues, different
genotypes, or at various developmental stages (Aharoni & Vorst, 2002; see also
Chapter 8). This technology can help identify genes that are up- or downregulated with a mineral process of interest, and these genes then can be critically
analyzed through various bioinformatic or functional strategies, as noted above.
For success in this approach, the key is to have microarrays with good coverage
of the transcriptome (including genes with no known function), and tissues that
demonstrate broad diversity for the mineral process of interest. This is where
characterized diversity among unique genotypes is important, as it can provide
useful, contrasting experimental samples for mRNA extraction.
12.5.2 The path to improved cultivars
In the context of this chapter, it is important to remember that an improved cultivar pertains not only to mineral density (or bioavailability), but to an improved
mineral trait in combination with the maintenance of other desired qualities (i.e.,
high yield, disease resistance). Thus, the full impact of nutritional genomics
only will be realized when its tools are integrated with conventional breeding
methodologies to generate new genotypes, stacked with several useful traits.
Gene identification is important to this process, because it provides very
direct and targeted tools for marker-assisted selection (MAS) in conventional
breeding. MAS usually is performed with molecular markers that are only linked
to a relevant QTL (as noted in Fig. 12.3). In other words, the marker helps
identify progeny that carry one parent’s locus, but this is done without specific
knowledge of the genes at that locus (Gepts, 2002). During the advancement
of breeding generations, any recombination that occurs between the molecular
marker and the actual allele conferring the improved trait will invalidate the
marker as a selection tool in subsequent generations. The further the marker is
from the relevant gene, the higher the probability that it can be separated from its
original allele by recombination. While MAS has been used successfully (Hash
et al., 2003), specific knowledge of the critical gene for a mineral process can
facilitate the development of gene-targeted selection approaches that are much
more robust (Andersen & Lübberstedt, 2003). Selection for molecular markers
that reside within a gene/allele of interest should allow an absolute maintenance
of that allele as generations are carried forward.
Allele discovery, therefore, plays a central role in facilitating gene-targeted
selection. Once a critical gene has been identified, specific primers can be developed to clone genomic segments from two diverse genotypes (i.e. that show
differences in mineral concentration). Sequencing of genomic DNA would lead
to the identification of single-nucleotide polymorphisms (SNPs) within introns,
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or length variations within exons, that could be exploited to develop specific
molecular markers for the allele of interest (Borevitz & Chory, 2004). Progeny
carrying the allele then would need to be assessed for mineral concentration, to
confirm the allele’s benefit.
In addition to its impact on conventional breeding, gene discovery, of course,
is essential for any genetic engineering approach using transformation technologies. Selected alleles of a mineral-related gene can be moved from one species to
another; thus, nutritional genomics research in model systems (e.g. Arabidopsis,
M. truncatula, rice) will play an important role in providing these genes. Once
identified, any number of strategies can be employed to express a transgene in a
crop of interest. Ubiquitous, inducible, tissue-specific, or development-specific
expression can be driven by appropriate promoters (Tomsett et al., 2004). The
latter require knowledge of genes and genome regulation that might be broader
than those pertaining to mineral physiology alone (e.g. a promoter region for
a seed storage protein could be used to drive seed-specific expression of other
genes; Lucca et al., 2001). Thankfully, there is much basic research being directed towards gene regulation and promoter discovery that the mineral scientist
can draw from (Shah et al., 2003; Qu & Takaiwa, 2004).
12.6 Future needs
Although the steps presented in Fig. 12.3 can be viewed in the context of a
single crop, we hope the preceding discussion has demonstrated that the genomic
resources of several species (crop and model plants; prokaryotic organisms) can
(and should) be applied to the goal of crop mineral improvement. Because of the
explosion of genome projects, and the continuing advances in technologies to
study whole genomes, we are optimistic that additional mineral-dense cultivars
are on the horizon, and that nutritional genomic research will play a significant
role in this effort. There are, however, several issues that need to be addressed
as these improvement efforts move forward, and we hope researchers will give
them due attention.
r For each mineral-crop situation, assessments should be made about the
need for upper limits to an improvement strategy. Some minerals are toxic
in excess, and thus projected daily intakes of a crop, in conjunction with
bioavailability percentage and mineral concentration must be integrated to
determine whether a problem might occur. Interdisciplinary collaboration
between plant scientists and human or animal nutritionists is encouraged
to address this issue.
r More information is needed on mineral distribution and storage within a
plant, to assess the capacity of different tissues to serve as repositories of
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minerals prior to subsequent partitioning. Knowledge of possible temporal
changes in these compartments is also required.
r For seed mineral improvement, a better understanding of source–sink
photoassimilate partitioning is needed in individual crops. Because most
minerals contribute minimally to phloem turgor pressure, they will be
carried along with the predominant flow of carbohydrates from wherever
they enter the pathway. Mineral remobilization thus may be highest from
strong source regions, such as flag leaves in cereals.
r For transgenic strategies, more information is needed on tissue-specific
or development-specific promoters, especially in leaf tissues that might
serve as sources of minerals for mobilization to seeds. When seed mineral
density is the ultimate goal, seed-specific transgene expression will not
necessarily impact mineral transport to that sink.
r For any mineral improvement effort, changes in the level of other minerals
must be monitored to ensure that required minerals are not lowered, and
toxic minerals are not accumulated.
Disclaimer
The contents of this publication do not necessarily reflect the views or policies of
the US Department of Agriculture, nor does mention of trade names, commercial
products, or organizations imply endorsement by the US Government.
Acknowledgements
This writing of this chapter was funded in part by the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number
58-6250-1-001.
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