Zinc plays critical role in

PLANT GROWTH
PROF ISMAIL CAKMAK, SABANCI UNIVERSITY, FACULTY OF ENGINEERING AND NATURAL SCIENCES, ISTANBUL, TURKEY

Zinc (Zn) is an essential micronutrient element and has a number of crucial functions in plant systems. Its deficiency in crop plants results in significant decreases in both productivity and nutritional quality. Under very severe zinc deficient soil conditions a complete reduction in crop yield may also occur. A concentration of diethylene triamine penta-acetic acid (DTPA)-exctractable Zn is a good indicator of the severity of zinc deficiency in soils. Figure 1 shows the growth of maize plants in a Zinc deficient soil with very low plant available Zn concentration (DTPA-Zn: around 0,1 mg/kg soil). Maize plants were grown in soil with (+Zn: 50 kg ZnSO4,7H2O per ha) and without (-Zn) a Zn application. The results indicated a very high susceptibility of maize to soil with a Zndeficiency. As also found in maize, typical morphological alterations developed in zinc deficient plants which include interveinal chlorosis on leaves, little leaf formation and reduced shoot elongation (stunt growth). Zinc deficiency is also a critical micronutrient deficiency in human beings in the developing countries, leading to severe impairments in growth, brain function and immune function. Zinc deficiency has been shown to be one of

the major reasons for child deaths worldwide. It is responsible for nearly 450 000 deaths in children under five years of age which is 4,4% of worldwide deaths in children under five (Black et al., 2008, Lancet 371:243 - 260). Due to their high requirement for Zn, children are very sensitive to a low dietary intake of Zn. Monotonous consumption of cereal-based foods as a major source of daily calorie intake has been shown to be a key reason for high prevalence of Zn deficiency in developing countries (Cakmak et al., 2008, Plant and Soil 302:1 - 17). Cereal grains are inherently very low in Zn concentrations. Growing cereal crops in soil with low concentrations of zinc further decreases grain Zn concentration. It is, therefore, not surprising that the well-documented zinc deficiency problem in human beings occurs predominantly in countries such as India, China, Pakistan and Turkey where soil are low in available Zn and where cereals are the major source of calorie intake (Figure 2).

chemical factors, such as high levels of calcium carbonate, pH and metal oxides and low levels of organic matter and soil moisture. Also in acid sandy soil, the available amounts of Zn are reduced because of the high potential for Zn leaching and lime applications. Among the soil factors affecting the solubility of Zn, soil pH plays a decisive role. For example, increasing soil pH from 5,5 to 7 reduces the solubility of Zn in soil up to 45fold. A sudden increase in soil pH through liming is therefore associated with large decreases in soluble Zn and consequently low amounts of Zn in plants. It is therefore important to monitor the Zn nutrition of crops after the soil has been limed.

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Functions of zinc in plant cells

Zinc has a number of fundamental functions in plant cells and therefore many physiological processes are adversely affected when plants are exposed to a Zn deficiency. When leaf or shoot zinc concentrations are below 15 mg/kg tissue, plants show very quick reductions in growth and a severe decline in yield. Under very severe zinc deficient soil conditions (DTPA-Zn: around 0,1 mg/kg), leaf Zn concentrations can even be below 10 mg/kg. Such low leaf concentrations of Zn are expected to be responsible for the loss of physiological functions of Zn and consequently very severe impairments in plant growth as shown in Figure 3 for wheat.

Soil zinc deficiency

It is widely believed that a Zn deficiency is the most widespread micronutrient deficiency in agricultural soil. It is estimated that nearly half of the cultivated soil globally contain low amounts of soluble Zn (Figure 2). In most cases, soil contains fairly high amounts of Zn but the solubility of Zn existing in soil is substantially low due to various soil physical and

Figure 1: Growth of maize plants with (+Zn: 50 kg ZnSO4.7H2O per ha) and without (-Zn) a soil zinc application on zinc deficient soil (DTPA-Zn: around 0,1 mg/kg soil) in the research farm of Bahri Dagdas International Agricultural Institute in Konya, Central Anatolia.

Figure 2: Overlap between geographical regions with soil zinc deficiency (red: widespread deficiency; green: medium deficiency) and high incidence of Zn deficiency in human populations.

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The most important functions of Zn in plant cells are: Protein synthesis and function; Structural and functional integrity of cell membranes; Detoxification of reactive oxygen species (ROS); Photo-oxidation and light damage; Synthesis and protection of IAA; and Reducing cadmium accumulation in plants.

Structural and functional integrity of cell membranes

The structural and functional integrity of proteins and proper functioning of biological membranes are under the control of Zn. Zinc greatly affects the architecture and controls the permeability of biological membranes. According to Welch et al. (1982, In: Proc. Ninth Plant Nutr. Collq. Warwick, UK, pp. 710 - 715), to fulfil this action, Zn must be present continuously in the external medium during growth. Biological membranes need Zn at very high concentrations. For example, Zn concentrations in animal cells vary from less than 1 nM in cytoplasm to higher than 1 nM in membrane vesicles (Cakmak, 2000, New Phytol. 146:185 - 205). When Zn is deficient, the structural integrity of membranes is impaired resulting in degenerative changes in membrane function. One of the consequences of the membrane damage is the alterations in uptake and transport of mineral nutrients. The effect of zinc on membrane function might be very important in saline soil with high concentrations of Na and boron. Zinc deficiency is a very characteristic micronutrient deficiency problem in semi-arid regions where salt accumulation is also a common problem in soil. Loss of structural integrity of root cell membranes under zinc deficiency may induce a high root uptake and high shoot accumulation of Na, leading to salt damage and an ion imbalance in plants. Improving the zinc nutritional status of plants grown on saline soil conditions is therefore an important task to minimise excess salt accumulation in plants.

in terms of root infection by pathogenic micro organisms. Under zinc deficiency, cell membranes become leaky and therefore, root exudation of various organic compounds (e.g. carbohydrates and amino acids) into soil (rhizosphere) is increased. As shown in Table 1, a Zn deficiency in different crop plants significantly enhanced root exudation of carbohydrates, amino acids and phenolic compounds. Such root exudates are excellent feeding substrates for pathogens and stimulates the fungal and bacterial invasion of roots. There is evidence indicating that zinc deficient plants are highly susceptible to pathogenic infection such as Fusarium graminearum, Gaumannomyces graminis and Rhizoctonia solani, most probably due to leaky membranes.

Protein synthesis and function

One of the well-documented functions of Zn is its role in protein synthesis and its contribution to the structural integrity of a number of proteins. Due to impairments in the structural integrity of ribosomes (sites of protein synthesis) and activities of the enzymes which are involved in protein synthesis (e.g. RNA polymerase) Zn deficiency results in a severe decline in protein biosynthesis (Marschner, 1995, Mineral Nutrition of Higher Plants, Academic Press). Conse quently, the amount and activity of various enzymes and zinc finger proteins (DNA-binding proteins) are seriously affected under Zn deficient conditions. Zinc finger proteins affect a number of fundamental cellular processes and are required for the expression and regulation of genes. Among all the micronutrients, Zn is needed by the largest number of proteins for their functions and structural integrity. Up to 10% of the proteins in biological systems need Zn for their proper functioning and for the maintenance of their structural integrity (Andreini et al., 2006, J. Proeteome Res. 5:3173 - 3178). It is, therefore, not surprising why plants show a high susceptibility to low concentrations of Zn in tissues (Figure 3).

Detoxification of reactive oxygen species (ROS)

Reactive oxygen species (ROS), such as superoxide radical (O2.-) and hydroxyl radical (OH.), are the major determinants of the cell damage under various stress factors, such as drought, low temperature stress and heat. ROS are produced by transferring electrons or energy to molecular oxygen and responsible for oxidative damage of chlorophyll, proteins, biological membranes and DNA. There are a number of enzyme systems or metabolic processes in plant cells which generate ROS by transferring electrons onto oxygen, particularly during photosynthetic or mitochondrial electron transport (Asada, 1999; Ann. Rev. Plant Physiol. Plant Mol Biol. 50:601 - 639). Most of the environmental stress factors (such as drought or chilling) cause cell damage by producing ROS. Among ROS, superoxide radical (O2.-) is very important because
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Pathogenic infection
The loss of membrane integrity and increases in membrane permeability are also important

Figure 3: Growth of wheat plants with (+Zn: 50 kg ZnSO4.7H2O per ha) and without (-Zn) soil Zn application in a zinc deficient soil (DTPA-Zn: around 0,1 mg/kg soil) at the research farm of Anatolian Agricultural Research Institute in Eskisehir.

Figure 4: The role of Zn deficiency in a generation of toxic oxygen free radicals and the effects of free radicals on plant growth (see Cakmak, 2000, New Phytol. 146:185 - 205).

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O2.- is responsible for the generation of other toxic O 2 species. Superoxide dismutase (SOD) is the enzyme which detoxifies superoxide radicals and plays a key role in the defence systems of biological systems against ROS. SOD is a Zn-dependent enzyme. There are three types of SOD enzymes and the Zn-containing SOD is the predominant form of SOD in both plant and mammalian cells. With a zinc deficiency, SOD activity is significantly depressed leading to a higher susceptibility of Zn deficient cells to an oxidative attack by ROS. Due to the reduced biosynthesis of proteins (see above), activity of other antioxidative enzymes is also reduced with a Zn deficiency which potentiates production of more ROS (see Figure 4). It is well-known that SOD has a critical protective role against ROS under various environmental stress factors such as drought, low temperature, salt and heat. Therefore, it can be suggested that plants with a low Zn nutritional status are more susceptible to environmental stress factors. There are several published data indicating that low temperature, stress, high light damage or drought stress in crop plants become more pronounced when plants are growing in Zn deficient soil conditions (Cakmak, 2000, New Phytol. 146:185 - 205). Superoxide generating NADPH-oxidase is a universal enzyme system which produces superoxide radicals in biological systems and represents an important source of ROS produced under different stress factors. The activity of this enzyme is very sensitive to a Zn supply. There is increasing evidence that shows that the activity of superoxide generating NADPH-oxidise is increased under Zn deficiency (Cakmak and Marschner, 1988, J. Exp.

Bot. 39:1 449 - 1 460). There is a very close relationship between cell damage (lipid peroxidation) and activity of superoxide generating NADPH-oxidase in different cell systems. Enhanced activity of superoxide generating NADPH-oxidase is another reason why a zinc deficiency makes plants highly susceptible to environmental stress factors (Figure 4). Improving the zinc nutritional status of plants is of great importance in alleviating cell damage caused by environmental stress factors. In semi-arid conditions with low rain and under high temperature, spike (ear) sterility is a very common problem in cereal crops. Studies in Central Anatolia (a region wellknown for its zinc deficiency) showed that a zinc deficiency problem exacerbates a spike sterility problem (Bagci et al., 2007, J Agron Crop Sci 193:198 - 206), probably due to increased amounts of ROS. A soil application of ZnSO4 was effective in reducing the severity of a spike sterility problem in wheat. Drought and heat stress, together with high light damage are well known to produce oxidative cell damage by producing ROS.

The transfer of electrons and light energy to O2 produces ROS and makes plants more sensitive to stress. As expected, a stressinduced ROS generation during photosynthetic electron transport is pronounced when plants grown under high light intensity (higher light intensity releases more electrons for O2 activation), leading to so-called photooxidative damage to chloroplasts. Zinc deficiency limits photosynthetic CO2 fixation due to the susceptibility of some photosynthetic enzymes to Zn deficiency and impairments in the utilisation of photoassimilates (Marschner and Cakmak, J. Plant Physiol. 134:308 - 315). The amount of nonutilised electrons and absorbed light energy is increasing in zinc deficient chloroplasts due to limited photosynthetic CO2 fixation. This situation in zinc deficient leaves activates the generation of ROS in chloroplasts. Due to low activity of the protective enzymes against ROS such as superoxide dismutase, ROS generation and oxidative damage (photoxidative damage) is additionally intensified with a zinc deficiency. Zinc deficient plants are, therefore, highly sensitive to high light conditions, especially in combination with an environmental stress factor. When exposed to high light intensity, Zn deficient plants very quickly develop leaf chlorosis and necrosis (Figures 5 and 6). A partial shading of the Zinc deficient plants strongly reduces the development of leaf chlorosis and further cell damage (Figure 5). It is, therefore, not surprising why zinc deficiency symptoms (leaf chlorosis) on perennials is more severe during the summer months and that more severe leaf chlorosis and necrosis develop on the sunny-sides of tress (Figure 6) or on branches that are directly exposed to high light intensity. Also

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Photo-oxidation and light damage

In plant cells, chloroplasts are major cell organelles producing ROS, especially during the photosynthetic electron transport. During photosynthesis, large amounts of electrons are produced for CO2 fixation, particularly under high light intensity. If photosynthetic CO2 fixation is reduced due to any environmental stress factors (e.g. drought), the electrons released and the light energy absorbed by pigments during photosynthesis are used intensively for the activation of oxygen instead of CO2 reduction.

Figure 5: The effect of partial shading on the development of leaf interveinal chlorosis in bean plants grown under low Zn supply (for further details, refer to Cakmak, 2000, New Phytol. 146:185 - 205).

Figure 6: The development of zinc deficiency chlorosis in grapefruit trees during winter time in a grapefruit orchard in South Turkey. Leaf chlorosis on branches was very severe on the sun-exposed (south-facing) sides. A zinc concentration of leaves varied between 7 mg to 9 mg/kg dry weight of leaf (for further details, refer to Cakmak et al., 1995, J. Plant Physiol. 146:355 - 360).

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these observations indicate that the maintenance of an adequate Zn nutritional status of plants under various environmental stress conditions is of great importance for the better survival and higher productivity of crop plants.

the nutritional quality and market value of foods. Cadmium is a toxic heavy metal and causes various health complications such as an increase in the risk of lung damage, kidney dysfunction and hypertension. Zinc and Cd are chemically very similar and thus they compete for the same or similar binding sites or channels to be absorbed and transported in root cells. Possibly, Zn and Cd also compete during phloem loading and translocation from leaves into seeds. Therefore, under zinc deficient soil conditions plants absorb and accumulate more Cd (Grant and Sheppard, 2008, Human Ecol. Risk Assess. 14:210 - 228). Consequently, high grain accumulation of Cd is very often reported in plants growing under low Zn supply, as shown in durum wheat and flax (Table 2). Soil zinc applications provide double benefits for human beings: low accumulation of Cd and high accumulation of zinc in seeds (Table 2). Adequate amounts of zinc also competitively inhibit phloem loading of Cd. The inhibitory effect of zinc on phloem transport of Cd from leaves into growing parts of plants (e.g. into seeds/grains) has been shown in a short-term experiment by using radiolabelled Cd (109Cd). The translocation of Cd from older leaves into other shoot parts of plants (e.g. phloem transport of Cd) was very clearly inhibited in plants with high zinc concentrations (Figure 7). This inhibitory effect of zinc on Cd transport in plants is very important to harvest grains with low Cd accumulation.

Biosynthesis and protection of phytohormone auxin

Auxin (IAA: Indole-3-acetic acid) is an important phytohormone produced mainly in meristematic sites and in the young expanding organs of plants. Cell elongation and expansion (and thus growth) is very much affected from a level of IAA. Increasing evidence is available showing that zinc is important for IAA biosynthesis and/or for protection of IAA from oxidative degradation by the attack of ROS. In zinc deficient plants the amount of IAA is reduced significantly and the application of Zn to Zn deficient plants increases and restores the levels of IAA again (Cakmak et al., 1989, 40:405 - 412). IAA is highly sensitive to oxidative degradation by ROS. By stimulating the production of ROS, Zn deficiency induces IAA oxidation in plant cells. It is believed that that well-known characteristic symptoms of zinc deficiencies, such as small leaves (reduced leaf size) and stunted growth (Figure 3) are ascribed to low levels of IAA.

yield and healthy food production. Today, nearly half of the cultivated soil has low amounts of plant available (soluble) zinc that affects both crop production and the nutritional quality of food crops for human beings. Plant requirements for zinc are increasing when plants are exposed to environmental stress conditions such as drought and heat. Zinc deficiency exacerbates adverse effects of environmental stress factors on plant growth due to intensified production and reduced detoxification of highly toxic oxygen radicals (ROS). It is, therefore, important to maintain a higher zinc nutritional status of plants when grown under stress conditions. Applications of zinc containing fertilisers would be a very useful strategy to keep readily available zinc in soil during plant growth in order to ensure healthy root growth and to maintain adequate root zinc absorption from soil. The enrichment of commonly applied NP or NPK fertilisers represents a quick and simple solution to contribute to a better zinc nutrition of crop plants and higher dietary intake of zinc in human populations. For the last 15 years zinc containing NP and NPK fertilisers are being applied to soil in Turkey. The amount of zinc added to NP and NPK fertilisers in Turkey is 1% pure zinc in the form of ZnSO4 or ZnO (Cakmak, 2008, Plant and Soil, 302:1 - 17). The economic benefits associated with the application of zinc fertilisers in Turkey are very impressive (at least $US 100 M per year estimated by the Ministry of Agriculture and CIMMYT-Ankara). In certain areas with extremely loz plant availability of Zn in soil (e.g., DTPA-Zn: 0,1 mg/kg soil) cereal production was not economic with grain yields of 250 kg/ha and Zn application has enhanced grain yield by a factor of 6 to 8 to
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Cadmium accumulation in food crops

Zinc nutrition of plants is also important for low cadmium (Cd) accumulation in the edible parts of plants. The accumulation of Cd in food crops is an increasing concern, especially in cereal grains and tubers that affects

Enrichment fertilisers with zinc

As indicated above with different examples, zinc is a critical micronutrient for a better

Figure 7: Inhibition of 109Cd translocation from older leaves into other parts of shoot in plants having high zinc concentrations (Source: Cakmak et al. 2000: Plant and Soil, 219:279 - 284).

Figure 8: Response of barley plants to foliar Zn application (0,5 % ZnSO4.7H2O) on a Zn-deficient soil in Central Anatolia (DTPA-Zn: 0,1 mg/kg soil). Source of picture: Cakmak et al., Plant and Soil, 180: 165-172.

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around 2 000 kg/ha. Figure 8 shows response of barley plants to foliar Zn application on a soil containing DTPA-Zn: 0,1 mg/kg soil. Besides such impressive economic benefits, increases in grain zinc concentration by zinc fertilisation also contribute to the daily zinc intake of people in Turkey and thus improving human health.

Similar to Turkey, in South Africa high amounts of zinc containing fertilisers are also applied. To our knowledge, 0,5% zinc containing NP or NPK fertilisers have been applied in South Africa for the past 25 years. According to current information, the total amount of zinc enriched fertilisers applied annually in South Africa and Turkey is about

500 000 and 400 000 tons, respectively. With the application of zinc fertilisers to soil particular attention should, however, be paid to the possible accumulation of zinc at excessive levels in soil and plants. Based on the long-term experiences in Turkey and South Africa, zinc toxicity in plants due to long-term application of zinc containing NP or NPK fertilisers is not observed and reported. However, if zinc is applied to soil only in the form of ZnSO4 or ZnO (without adding it to NP or NPK fertilisers), there is no need to apply ZnSO4 or ZnO every year. According to long-term experiments, zinc applied in the form of ZnSO4 up to 100 ZnSO4 kg per ha is effective for two to three years. For enquiries contact Ismail Cakmak at cakmak@sabanciuniv.edu.

TABLE 1: CHANGES IN ROOT EXUDATION OF AMINO ACIDS, SUGARS AND PHENOLICS IN COTTON, WHEAT AND APPLE PLANTS GROWN IN A NUTRIENT SOLUTION WITH A DEFICIENT (-ZN) AND ADEQUATE (+ZN= 1 M) ZN SUPPLY (CAKMAK AND MARSCHNER, 1988, J. PLANT PHYSIOL. 132:356 - 361).

ZN Treatment -Zn +Zn -Zn +Zn -Zn +Zn

Amino Acids 165 48 48 21 55 12

Sugars (g g -1 root 6h -1) Cotton 751 375 Wheat 615 315 Apple 823 275

Phenolics 161 117 80 34 350 103

Acknowledgement
The author is grateful to BD International Agricultural Research Institute in Konya and Anatolian Agricultural Research Institute in Eskisehir for along term successful collaboration in zinc research in Central Anatolia.

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TABLE 2: THE EFFECT OF SOIL ZINC APPLICATION ON CONCENTRATIONS OF ZINC AND CD IN FLAX SEED AND DURUM WHEAT GRAIN. SOURCE: JIAO ET AL., 2004: J. SCI. FOOD CHEM. 84:777 - 785.

Fertiliser treatments * MAP MAP MAP+Cd MAP+Cd TSP TSP

Zinc fertilization No Yes No Yes No Yes

Grain Zn concentration Flax Durum wheat (mg Zn/kg) 22 9 39 27 25 10 42 31 22 8 41 27

Grain Cd concentration Flax Durum wheat (g/kg) 226 267 136 90 330 275 211 165 228 254 138 112

*: MAP: Monoammonium Phosphate; MAP+Cd: Monoammonium Phosphate containing Cd; TSP: Triple Superphosphate.