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Zinc is essential for all organisms. It is a group II b metal, with a completed d subshell and two additional s electrons. Thus, unlike other cationic micronutrients, it has only one oxidation state (Zn2+). Zinc plays a structural as well as functional role in plants. It forms a structural component of a large number of proteins with catalytic or regulatory functions. Usually, the Zn2+ ion binds to nitrogen or sulphur-containing ligands through ionic bonds, forming a tetrahedral geometry. Over three hundred enzymes are known to contain Zn as a cofactor (Valee and Auld, 1990). By providing stability to many regulatory proteins, or domains thereof (zinc fingers, zinc clusters and RING finger domains), zinc plays a role in transcriptional regulation (Coleman, 1992). Zinc may chelate with polypeptides, synthesized in response to excessive accumulation of heavy metals (including itself), to form metallopolypeptides like phytochelatins and metallothioneins and contribute to tolerance mechanisms against metal hyperaccumulation. Over the years, zinc has been shown to play critical roles in plant reproductive development, prevention of water stress and protection against toxic effects of reactive oxygen species.


Zinc is largely taken up by plants in the ionic form, as free Zn2+. Studies based on root uptake of Zn2+ by certain graminaceous species grown over a wide range of Zn concentrations in solution cultures (Chaudhry and Loneragan, 1978; Veltrup, 1978; Mullins and Sommers, 1986) showed Zn2+ uptake to be concentration-dependent and saturable, which implied its carrier-mediated transport. Measurement of Zn2+ influx from chelated buffered solutions, in which Zn2+ activity was maintained close to the concentrations found in soil solution in most agricultural soils, lent support to carrier-mediated transport of Zn2+ (Norvell and Welch, 1993; Hart et al. 1998). Hart et al. (1998) showed Zn2+ uptake by wheat seedlings to be concentration dependent, with smooth saturating curves characteristics of carrier-mediated transport. Uptake was also shown to be inhibited by low temperatures indicating dependence on active metabolism. Zinc uptake measurements, using the Chara corellina model (Reid et al. 1996) showed a bi-phasic pattern of uptake, which showed that Zn2+ uptake was mediated by two separate systems—a high-affinity system that saturated at 100 nm and a low-affinity system that showed a linear dependence on concentrations upto at least 50 mM. Strong evidence in support of involvement of both a low concentration-high affinity system and a high concentration-low affinity system in Zn2+ uptake has come from short-term measurement of 65Zn2+uptake by wheat cultivars grown hydroponically at zinc concentrations ranging from 0.1 nM to 80 mM (Hakisalhoglu et al. 2001). According to Hakisalihoglu et al. (2001), the high-affinity transport system forms the predominant mechanism for Zn2+ uptake from soils, in which Zn2+concentration in the soil solution lies close to the lower limits of the nanomolor range (Welch, 1995).

Phytosiderophores, involved in iron uptake by graminaceous plants, are also secreted by the plants in response to Zn-deficiency (Zhang et al. 1989, 1991a; Walter et al. 1994; Cakmak et al. 1994, 1996; Rengel 1997; Rengel et al. 1998; Hopkins, 1998). Reported ability of phytosidophores to mobilize not only iron but also zinc from calcareous soils (Treebly et al. 1989) suggested their possible involvement in mobilization and uptake of zinc under Zn-deficiency conditions. Evidence for a role of phytosiderophores in enhancing Zn mobilization and uptake came for the studies reported by Zhang (1999a) and Von Wiren et al. (1996) and reports of a positive relationship between the ability of wheat genotypes to secrete phytosiderophores and their zinc efficiency (Walter et al. 1994; Cakmak et al. 1994,1996). Hopkins et al. (1998) attributed the differences in tolerance of sorghum, wheat and corn to Zn deficiency to the ability of their roots to release phytosiderophores under conditions of zinc deficiency. When Zn supply was limiting, release of phytosiderophores by roots followed the order wheat> sorghum >corn, which corresponded to their tolerance to Zn deficiency (Hopkins et al. 1998). Doubts have, however, been raised about release of phytosiderophores by roots of graminaceous plants as a mechanism for enhanced mobilization and uptake of Zn in response to Zn deficiency. Erenoglu et al. (1996) did not find any positive relationship between Zn efficiency of bread wheat genotypes and the capacity of their roots to secrete phytosiderophores. According to Rengel (1998), enhanced secretion of phytosiderophores under Zn deficiency is possibly caused by Zn-deficiency induced impairment in iron transport resulting in its deficiency, which activates the secretion of the phytosiderophores.

Several transporters belonging to the ZIP (ZRT-IRT-like proteins) (Guerinot, 2000) and the CDF (cation diffusion facilitator) family (Williams et al. 2000) have been recently suggested to be involved in transport of zinc. Four ZIP transporter genes, ZIP1, 2, 3 and 4, have been isolated from Arabidopsis (Grotz et al. 1998; Guerinot, 2000). ZIPl, 2,3 and 4 are expressed in roots of zinc-deficient plants. ZIP4 is also expressed in shoots and is predicted to have a chloroplast-targeting sequence. Expression of ZIPl, Z/P2, and ZIP3 genes of Arabidopsis in zinc-defective yeast mutant zrtl zrtl has been shown to restore growth limitation due to zinc-deficiency. Another zinc transporter gene ZNT1 has been cloned from Zn/Cd accumulator plant Thalspi caerulescens (Lasat et al. 2000; Assuncao et al. 2001). The Tc ZNT1 is a ZIP gene homologue and expressed at high levels in both roots and shoots (Assuncao et al. 2001). In the non-hyperaccumulator species T.arvense, TcZNTl, and another ZIP gene homologoue ZNT2, are expressed at much lower levels and only in response to zinc deficiency (Lasat et al. 2000; Assuncao et al. 2001). Another member of the ZIP family Gm ZIPl, isolated from soybean, has recently been shown to functionally complement the zrtl/zrt2 mutant of yeast (Moreau et al. 2002). It has been reported that Gm ZIPl is highly selective for zinc and is expressed only in root nodules, where it is largely localized to the peribacterial membrane, suggesting its possible role in symbiosis (Moreau et al. 2002).

Van der Zaal et al. (1999) have characterized a cation diffusion facilitator family transporter ZAT from Arabidopsis and showed that it functions in zinc transport under condition of zinc excess. At ZAT is constitutively expressed throughout the plant. Its overexpression in transgenics exposed to zinc excess leads to increased accumulation of zinc. Van der Zaal et al. (1999) have suggested that ZAT-mediated transport of zinc leads to vascular/vacuolar sequestration of zinc, contributing to zinc homeostasis and tolerance.

Zinc is phloem mobile. Haslett et al. (2001) have shown that foliar application of zinc to wheat can provide sufficient zinc for meeting the requirements for vigourous vegetative growth. Zinc, supplied through leaves, was shown to be phloem translocated to different plant parts including roots. In wheat, sufficient amount of zinc (65Zn) has been shown to be translocated from leaves to the developing grains during the grain-filling period (Pearson and Rengel, 1994, 1996; Pearson et al. 1995). Transport to seeds has been shown to be influenced by zinc status of the mother plants (Pearson and Rengel, 1995a). Limitation in zinc transport under conditions of zinc deficiency could result from its possible binding to cellular metabolites, Zn enzymes and the exterior of the plasma membrane (Welch, 1995).

Phloem transport of Zn takes place in the form of zinc complexes with organic acids such as citric and malic acids (White, 1981a,b) or nicotianamine (Stephan and Scholz, 1993; Schmidke and Stephan, 1995) Recent studies by Takahashi et al. (2003) suggest a role of nicotianamine in transport of zinc to floral parts and intracellular trafficking to sites where it is integrated in to the functional proteins.

5.3 ROLE IN PLANTS 5.3.1. Enzyme Action

Zinc is a constituent of a multitude of enzymes (Valee and Auld, 1990; Valee and Falchuk, 1993; Berg and Shi, 1996). Since zinc has only one oxidation state (Zn2)—unlike other metalloenzymes—Zn enzymes cannot function in oxidation-reduction reactions. In the Zn metalloproteins, the zinc ion binds to three-imidazole groups (Schiffs' base), with the fourth coordination position left free for interacting with the substrate. Generally, the fourth coordination site is bound to a water molecule that facilitates hydration or hydrolysis reactions and most zinc enzymes catalyse such reactions.

Carbonic Anhydrase (EC

Carbonic anhydrase (CA) contains zinc as an essential cofactor and is found in abundance in all living beings. Unlike the mammalian enzyme, which is a monomer, the plant enzyme can be a dimer, tetramer, hexamer or an octamer. Each subunit contains a single zinc atom, bound close to the active site to three imidazole rings of the histidine residues. The fourth coordination site is left free to react with the substrate. The enzyme catalyzes the reversible conversion of carbon dioxide to bicarbonate, which has high solubility in water.

Carbonic anhydrase is critical to photosynthesis in C4 plants, in which the first carboxylation reaction is catalyzed by phosphoeno/pyruvate carboxylase (PEPCase), which uses bicarbonate as a substrate (O'Leary, 1982). Unlike the C3 plants, wherein CA is largely localized to mesophyll chloroplasts, in C4 plants CA is localized to the cytosol of the mesophyll cells, which is also the site for PEPCase-catalyzed carboxylation (Burnell and Hatch, 1988). An analysis of the CA activity (at in vitro C02 concentrations) and associated rates of photosynthesis, showed that even a small decrease in CA activity could result in such a large decrease in steady-state HC03~ concentration, as could limit C4 photosynthesis (Hatch and Burnell, 1990). In C3 plants, the role of CA in photosynthesis has been somewhat uncertain (Utsunomiya and Muto, 1993). Randall and Bouma (1973) observed decreased CA activity with little effect on photosynthesis in zinc-deficient plants. Inhibition of CA activity did not cause a decrease in photosynthesis (Sasaki et al. 1998).

Superoxide Dismutase (EC

The enzyme structure and catalytic function have been described in Chapter 4 (Section 4.3.1.).

Alcohol Dehydrogenase (EC

Alcohol dehydrogenase contains two zinc atoms per molecule, one of which performs a structural and the other a catalytic function (Coleman, 1992) Alcohol dehydrogenase catalyzes the oxidation of acetaldehyde, formed by decarboxylation of PEP generated during glycolysis, to ethanol.

Acetaldehyde + NADH - Ethanol + NAD+

Increased activity of alcohol dehydrogenase in response to anaerobic stress such as flooding enables the plant roots/tissues to temporarily meet the energy requirement from ethanolic fermentation (Gibbs and Greenway, 2003; Ravichandran and Pathmanabhan, 2004). Carboxypeftidase A (EC

Carboxypeptidase A is a zinc metalloenzyme that catalyzes the hydrolysis of peptide bond at the C-terminal end by activating a water molecule.

Peptidyl-L-amino acid + H20 ^ Peptide + amino acid Carboxypeptide A is widely distributed. The plant enzyme catalyzes the hydrolysis of reserve proteins of seeds. Other Zinc Enzymes

Zinc is a cofactor of certain DNA-dependent RNA polymerases involved in transcription (Falchuk et al. 1977; Petranyl et al. 1978). Zinc is tightly bound to the enzyme protein and its removal results in enzyme inactivation. Petranyl et al. (1978) reported inactivation of wheat germ RNA polymerase II on removal of Zn from the enzyme protein. Strater et al. (1995) have described the crystal structure of a purple acid phosphatase from kidney bean which contains a dinuclear Fe (Ill)-Zn(II) active site.

There are several enzymes that are known to have a cofactor requirement of zinc in animals, fungi and bacteria but not in plants. Some of these are of wide occurrence in plants and their activities are inhibited under zinc deficiency. Common examples of such enzymes and the reactions catalyzed by them are:

NAD*-Glutamate Dehydrogenase (EC

L-Glutamate + NAD+ ^ a-Ketoglutarate + NH4+ + NADH + H+

Fructose-l,6-bisphosphate ^ Dihydroxy acetone phosphate +


ALA Dehydratase (EC4.2.1.24)

8-Aminolevulinate v Porphobilinogen


A plasma membrane H+-ATPase of corn roots has been shown to use zinc as the substrate and function as Zn-ATPase (Katrup et al. 1996).

5.3.2 Zinc Finger Proteins

Apart from being a constituent of zinc-metalloenzymes, zinc functions as a constituent of several regulatory proteins which interact with DNA and control gene expression. In the form of zinc finger, it forms a structural motif of the DNA-binding region of the transcriptional regulatory proteins (Berg and Shi, 1996). Originally recognized in transcription factor TF III A, involved in the transcription of 5s RNA by RNA Polymerase III genes in Xenopus, zinc finger motifs have been identified for several other transcription factors (Klug and Rhodes, 1987). The TF IIIA-type DNA binding proteins, also designated as the C2H2 or the classical zinc finger proteins, contain a Zn atom which is tetrahedrally coordinated to two Cys and two His residues to form a compact finger structure (Fig. 5.1). Structural studies over the past decade have shown that the TF IIIA-type Zn finger proteins of plants have two characteristic features, which distinguish them from other eukaryotic zinc figure proteins. First, the a - helix of the zinc finger, which makes contact with the major genome of DNA, contains a conserved amino acid sequence QALGGH; second, the adjacent fingers are separated by long spacers of diverse lengths, which recognize the spacing between the core sites of the target DNA (Takatsuji, 1999). The number of zinc fingers in a TF IIIA-type zinc finger protein range from one to four, with each additional finger resulting in increased specificity (Takatsuji, 1999; Laity et al. 2001). Some TF IIIA-type zinc finger proteins are involved both in protein-protein and protein-DNA interactions (Mackay and Crossby, 1988). While some zinc fingers interact with proteins, others react with DNA. First to be identified in a DNA-binding protein of petunia (EPF, renamed ZPT2-1), (Takatsuji et al. 1992), several TF IIIA-type zinc finger proteins have been isolated and characterized from different plants species and shown to be implicated in floral organogenesis, leaf initiation, gametogenesis and stress responses (Takatsuji, 1999). The gene encoding a TF IIIA-type zinc finger protein SUPERMAN (SUP), isolated and characterized from Arabidopsis has been shown to control the cell division demarcating the boundary between the third and the fourth whorl of flowers and determine the stamen number and carpel development (Sakai et al. 1995). It is also involved in morphogenesis (Gaiser et al. 1995). Nakagwara et al. (2004) have described a SUP gene homologue from petunia (Ph SVP1), whose product also functions in morphogenesis of placenta, anther and connective tissues between the floral organs. More recently, Nakagwara et al. (2005) have described another zinc finger protein from petunia which structurally resembles SUP and designated it as LIF (for Lateral shoot-Inducing Factor).

The L1F cDNA has been shown to be specifically expressed around the dormant axillary buds of transgenic petunia plants. Its over expressions induces axillary buds and dwarfed growth. The alterations in plant forms are attributed to changes in the cytokinin metabolism. Nakagawa et aL (2005) haves suggested that controlled expression of LIF transgene can possibly be made use of in optimizing the extent and the pattern of shoot branching to increase the harvest yield of plants.

Fig 5.1. Schematic diagram of zinc finger motifs. Each unit shows a Zn;' ion tetrahed rally ltganded to two Cys and two His residues. (Reprinted, with permission, from Biochemistry. D Votrt, JG Voet, eds ©2004 )ohn Wiley & Sons, Inc).

Yanagisawa (1995) have characterized a family of single zinc finger proteins, named Dof (for DNA binding with one finger), from plants. In the Dof zinc finger protein, the zinc atom is coordinated to Cys residues only (C2C2), with the zinc finger domain containing a 52 amino acid stretch. The Dots are suggested to function as transcriptional activators or repressors of tissue-specific and light-regulated gene expressions in plants (Yanagisawa and Sheen, 1998).

Recently, transcription factors with zinc-finger binding domains have been designed and used for targeting specific DNA sequences of endogenous genes (Guan et al. 2002; Ordiz et al. 2002). Guan et al, (2002) designed a polydactyl zinc finger transcriptional factor that could target the Arabidiopsis gene A PETALS 3 (AP 3) and induce a dramatic change in its expression. Another important biotechnological use to which zinc finger proteins have been put is the designing of zinc finger nucleases and using them for targeted mutagenesis (Lloyd et al. 2005). Zinc finger nucleases, synthesized by fusing non-specific DNA cleavage domains from Fok 1 endonuclease with DNA-binding domains composed of three Cys-2 His-2 fingers (Kim et al. 1996), make effective tools for targetted mutagenesis of plant genes (Lloyd et al. 2005).

5.3.3 Membrane Integrity

Bettger and O'Dell (1981) showed the importance of zinc in maintaining the structural integrity of biomembranes. Welch et al. (1982) were the first to suggest zinc involvement in the permeability of plant plasma membranes. They showed enhanced leakage of 32P and 36C1 from roots of wheat plants subjected to zinc deficiency. Resupply of zinc showed reversal of enhancement in leakage of the ions. Welch et al. (1982) concluded that deficiency of zinc makes the plasma membranes more leaky due to loss of structural integrity. Lindsay et al. (1989) suggested that zinc is involved in protection of the plasma membrane at its apoplasmic side. They opined that zinc reacts with negatively charged molecules of the membranes and contributes to their structural stability. Cakmak and Marschner (1988a) showed enhanced leakage of solutes (K+, amino acids, sugars, phenolics) by roots of zinc-deficient plants of four different plant species, which could be reversed on resupply of zinc, a feature not shown by calcium that is known for its effect on membrane permeability. Plasma membrane vesicles isolated from roots of zinc-deficient plants also showed higher permeability (proton flux) than the vesicles isolated from roots of Zn-sufficient plants (Pinton et al. 1993).

Effect of zinc deficiency in increasing membrane permeability resembles the effect induced by enhanced peroxidation of the membrane constituents (Van Ginkel and Sevanian, 1994). Zinc-deficient plants show decreased fatty acid content of the membranes. Effect on the concentration of unsaturated fatty acids and phospholipids (Cakmak and Marschner, 1988c) and that on reactive sulphydryl (-SH) groups is particularly marked (Welch and Norvell, 1993; Rengel, 1995b). The decrease in the concentration of -SH groups under zinc deficiency is more in zinc-sensitive wheat cultivar Durati, than in tolerant cultivar Warigal, and is reversed on resupply of zinc (Rengel, 1995b). Zinc has been suggested to protect the -SH groups of plasma membrane proteins from oxidative damage.

5.3.4 Anti Oxidant Activity

There is increasing evidence for a protective role of zinc against oxidative stress (reviewed by Cakmak, 2000). Oxidative stress results from production of reactive oxygen species (ROS) in quantities in excess of what can be effectively detoxified by the plant's inherent antioxidant system. Zinc protects plants from oxidative damage in two ways: by preventing enhanced production of the ROS; and by their rapid detoxification. Zinc inhibits the accelerated production of ROS by inhibiting the activity of membrane bound NADPH oxidase, which catalyzes the production of superoxide ions.

NADPH oxidase

Bettger and O'Dell (1981) provided evidence of zinc involvement in protection of cellular membranes against peroxidative damage from NADPH-derived free radicals. Enhanced peroxidation of membrane lipids destabilizes the membranes and makes them more leaky.

As a constituent of superoxide dismutase (Cu, Zn SOD), a key component of the inherent antioxidant defense mechanism of plants, zinc catalyzes rapid detoxification of superoxide ions by producing hydrogen peroxide, which can be taken care of by the other enzymatic components of the antioxidant system (e.g. catalase, ascorbate peroxidase). Accumulation of superoxide ions (02~) may lead to production of hydroxyl (OH*) radicles (Haber-Weiss reaction), which are even more reactive than 02~ and cause more severe damage to cellular membranes, nucleic acids and proteins.

5.4 deficiency responses 5.4.1 Visible Symptoms

Zinc deficiency symptoms generally appear in subterminal leaves after apparently normal growth during the early stages. Zinc deficiency responses of plants are varied. Common symptoms, shared by most plants, include reduction in leaf size and condensation of shoot growth. Combination of the two is often reflected as the clustered or rosetted appearance of the terminal growths. Fading of lamina, intervenal chlorosis, often associated with development of reddish-brown or bronze tints, and a metallic sheen on leaves are other common symptoms of zinc-deficiency. In some crops, leaves of zinc-deficient plants exude a viscous fluid, which on drying, appears as salt incrustations on the leaf surfaces. Such a feature is possibly a result of damage caused to cell membranes (Section 5.3.3). Response of vegetable crops to zinc deficiency is varied. Beans and radish exhibit zinc deficiency in the form of intervenal chlorosis. The chlorotic leaf areas turn brown, papery and necrotic, ending up in disintegration of the lamina. Leaves of zinc-deficient plants of cabbage and cauliflower show thickening of laminae and a thick deposition of epicuticular wax. These leaves remain turgid, possibly because of restricted opening of stomata. Leaves of zinc-deficient tomato plants show epinasty and inward curling of lamina, resembling auxin deficiency response. The laminae also turn thick and brittle. Older leaves often develop brown or orange tints and turn necrotic.

Amongst the cereal food crops, maize, rice and wheat are extremely sensitive to zinc deficiency. Symptoms initiate in fully expanded young leaves as fading of lamina and appearance of light brown necrotic lesions some distance away from the base. With continued deficiency, the lesions coalesce and spread apically. The apical part of the leaves of Zn-deficient wheat plants wither out, curl, hang down and even break out from the middle of the leaf. Zn-deficient wheat leaves also show epidermal exudations suggestive of leaky membranes.

5.4.2 Enzyme Activities

Plants subjected to zinc deficiency display alterations in the activity of many enzymes, including those which are not known to have any specific requirement of zinc as a cofactor or activator. Decrease in carbonic anhydrase activity in leaves is an universal response to zinc deficiency (Wood and Sibly 1952; Bar-Akiva and Lavon, 1969; Edward and Mohamad, 1973; Sharma et al. 1981a, 1982; Seethambaram and Das 1985; Silverman 1991; Pandey and Sharma, 1998, 2000; Rengel, 1995; Sasaki et al. 1998; Pandey et al. 2002b). Resupply of zinc to Zn-starved plants reverts the enzyme activity to normal (Sharma et al. 1981a; Bisht et al. 2002b). While there is a correlation between zinc supply, leaf tissue concentration of zinc and carbonic anhydrase (CA) activity, for the same level of zinc content different genotypes may show substantial differences in CA activity. Rengel (1995a) described higher CA activity in Zn-efficient wheat genotype 'Warigal' than in Zn-inefficient wheat genotype 'Durati' even when their leaf tissue concentration of zinc was about the same. Zinc deficiency induced decrease in CA activity has been shown to be associated with a decrease in the stomatal opening (Sharma et al. 1995; Pandey and Sharma, 2000) and an increase in C02 transfer resistance (Pandey and Sharma, 1989; Sasaki et al. 1998). Sharma et al. (1995) showed a good correlation between zinc concentration, guard cell CA activity (and K+ content) and stomatal opening. Zinc-deficient rice plants are reported to show loss of alcohol dehydrogenase activity (Moore and Patrick, 1988).

Zinc-deficient plants also show decreased activity of superoxide dismutase (SOD) (Cakmak and Marschner, 1993; Yu et al. 1998; Yu and Rengel, 1999; Pandey et al. 2002a,b). As expected, compared to the total SOD activity, the activity of Cu-Zn SOD is more responsive to zinc deficiency (Yu and Rengel, 1999; Pandey et al. 2002a,b). Leaves of zinc-deficient plants, showing low Cu-Zn SOD activity, may exhibit elevated activity of Fe-SOD (Yu and Rengel, 1999 Bonnet et al. 2000). As in case of CA, genotypes may differ in their efficiency of zinc utilization in SOD activity. Cakmak et al. (1997c) showed genotypic differences in SOD activity for the same level of zinc deficiency. Activities of other enzymes of the antioxidative defense system are also influenced by Zn nutrient status (Fig. 5.2). Zinc-deficient plants show low activities of catalase (Marschner and Cakmak, 1989; Bisht et al. 2002b) and ascorbate peroxidase (Yu et al. 1998; Pandey et al. 2002b).

Glutathione reductase (GR), an important enzyme of the ascorbate-glutathione cycle, also responds to changes in Zn nutrient status of plants but the effects of Zn deficiency on GR activity are inconsistent. Cakmak and Marschner (1993) described a decrease in GR activity in zinc-deficient bean plants. On the contrary, Pandey et al. (2002b) reported increase in GR activity in leaves of zinc-deficient maize. Not much is known about the significance of enhancement in GR activity under zinc deficiency; but it is reported that increase in GR activity in response to xenobiotics and environmental stresses contributes to plant tolerance against these stresses (Malan et al. 1990; Lascano et al. 1998; Donahue et al. 1997).

There are several reports of zinc affecting the activities of non-zinc enzymes. Inadequate supply of zinc causes a decrease in the activity of ribulose biphosphate carboxylase (Jyung and Camp, 1976), fructose 1,6 biphosphatase (Shrotri et al. 1983) and fructose 1,6 biphosphate aldolase (O'Sulliuan, 1970; Agarwala et al. 1976; Sharma et al. 1981; Quinland and Miller, 1982; Pandey et al. 2002b). Bisht et al. (2000b) have reported a sixfold increase in alanine aminotransferase activity in leaves of tomato plants subjected to zinc deficiency. On recovery from zinc deficiency, the enzyme activity was restored to near normal.

Zinc is reported to exert a strong inhibitory effect on the plasma membrane-bound NADPH oxidase, which catalyzes the production of superoxide (0"2) ions. Cakmak and Marschner (1988b) reported increased activity of NADPH oxidase in roots of zinc-deficient cotton plants. Supplying zinc to the deficient plants reversed the effect. Pinton et al. (1994) reported marked enhancement in the NADPH oxidase activity, with concomitant increase in 0"2 production in plasma membrane vesicles isolated from zinc-deficient bean plants.

Several workers have reported the enhancement of ribonuclease (RNase) activity under zinc deficiency (Dwivedi and Takkar, 1974; Johnson and Simons, 1979; Sharma et al. 1981a; Chatterjee et al. 1998; Bisht et al. 2002b; Pandey et al. 2002b). Increase in RNase activity, even before the external manifestation of zinc deficiency (symptoms), prompted Dwivedi and Takkar (1974) to suggest RNase as a biochemical parameter for diagnosing zinc deficiency. The increase in RNase activity is related to the extent of the deficiency (Sharma et al. 1981a) and is reversed on raising Zn supply from deficiency to sufficiency level (Bisht et al. 2002b). Zinc deficiency induced activation of RNase is associated with a decrease in the total RNA concentration (Sharma et al. 1981a; Bisht et al. 2002b). These observations suggest a role of zinc in regulation of RNA metabolism. Possibly, zinc prevents RNA degradation through allosteric inhibition of RNase. Low cellular concentration of zinc negates its inhibitory action on the enzyme, leading to accelerated degradation of RNA and, consequently, a decrease in protein synthesis. Similar to RNase, acid phosphatases are also activated under Zn deficiency (Hewitt and Tatham, 1960; Sharma et al. 1981a; Bisht et al. 2002b; Pandey et al. 2002b). Activation of acid phosphatases under Zn deficiency is also reversed on recovery from deficiency. Zinc is possibly involved in the maintenance of organic pool of phosphorus.

Presently, there is lack of direct evidence to substantiate zinc regulation of non-zinc enzymes in higher plants, but in yeast and animal cells, a number of metabolically critical enzymes are known to be inhibited by zinc and activated on removal of the metal from the inhibiting site (Maret et al. 1999). It is not unlikely that zinc may influence the synthesis of enzymes with no specific Zn requirement by virtue of its role as a constituent of RNA polymerase and zinc finger transcription proteins.

5.4.3 Photosynthesis

Plants exposed to zinc deficiency show decreased rate of photosynthesis (Fujiwara and Tstsumi, 1962; Ohki, 1976; Ghildiyal et al. 1978; Shrotri et al. 1981; Pandey and Sharma, 1989; Hu and Sparks, 1991; Sharma et al. 1994). Zinc nutrition effect on photosynthesis may involve changes in chloroplast structure, photosynthetic electron transport and/or COz fixation. Zinc deficiency induces changes in chloroplast ultrastructure (Thomson and Weier, 1962; Shrotri et al. 1978). Leaves of zinc-deficient maize plants show abnormalities in both mesophyll and bundle sheath chloroplasts. While mesophyll chloroplasts show a decrease in the number of grana and size of the granal thylakoids, the bundle sheath chloroplasts show, increased inter lamellar spaces and presence of osmophyllic globules (Shrotri et al. 1978). Low rates of photosynthesis in zinc-deficient plants could also result from damage to the thylakoid membranes caused by enhanced generation of reactive oxygen species under zinc deficiency (Marschner and Cakmak, 1989; Cakmak and Engels, 1999). Henriques (2001) has shown that zinc deficiency causes disorganization of chloroplast thylakoids, followed by degeneration of thylakoid and stromal components, causing a corresponding decrease in the photosynthetically active area of leaves. Effect of zinc on photosynthesis may also involve inhibition of carbonic anhydrase (CA) activity (Badger and Price, 1996). In C4 plants, CA catalyzes the production of bicarbonate (HCO"3), which functions as the substrate of phosphoeno/pyruvate carboxylase (O'Leary, 1982; Hatch and Burnell, 1990). In C3 plants, CA contributes to a C02 concentration mechanism at the site of carboxylation by RuBP carboxylase by facilitating the conversion of HCO"3 to C02 (Cooper et al. 1969). This may become crucial under zinc deficiency, when there is a decrease in C02 concentration because of increased resistance to C02 diffusion following closure of stomata (Sharma et al. 1982, 1995c; Pandey and Sharma, 1989; Sasaki et al. 1998).

Sharma et al. (1995c) have suggested CA involvement in stomatal functioning, and this may compound the effect of zinc deficiency on photosynthesis.

Srivastava et al. (1997) showed decreased photosynthetic rate, C02 partitioning and essential oil accumulation in leaves of zinc-deficient peppermint. Incorporation of 14C02 in essential oil in young developing leaves, which are actively involved in oil biosynthesis, was also decreased. The authors inferred that inadequate supply of zinc limits the production of photoassimilates, as also their availability for essential oil biosynthesis. When zinc supply is not adequately met, the products of photosynthesis are possibly utilized in primary metabolism in preference to essential oil biosynthesis.

5.4.4 Carbohydrates

Leaves of zinc-deficient plants show increased accumulation of carbohydrates (Sharma et al. 1982; Marschner and Cakmak, 1989; Marschner et al. 1996). Sharma et al. (1982) showed marked accumulation of starch in Zn-starved leaves of cabbage. Two possible explanations have been advanced for increased accumulation of starch in Zn-deficient plants. The first explanation involves limitation in shoot-root partitioning of photoassimilates (Marschner and Cakmak, 1989), possibly because of a limitation in phloem loading. The second involves a limitation in development of adequate sink capacity resulting from reduced male fertility and seed set under zinc deficiency (Sharma et al. 1990). Zinc is also suggested to be involved in starch metabolism (Jyung et al. 1975) and sucrose biosynthesis (Shrotri et al. 1980).

5.4.5 Nucleic Acids, Proteins

There are several reports of increased accumulation of non protein nitrogen and decrease in protein content in leaves of zinc-deficient plants (Sharma et al. 1982; Kitagishi and Obata, 1986; Obata and Umebayashi, 1988; Obata et al. 1999; Cakmak et al. 1989; Bisht et al. 2002b). Obata and Umebayashi (1988) showed a requirement of zinc for synthesis of protein in cultured cells of tobacco. Decrease in protein content in response to Zn deficiency has been attributed to Zn deficiency induced changes in ribosomes and nucleic acid metabolism. Leaves of zinc-deficient plants show a decrease in concentration of ribonucleic acid (Sharma et al. 1982; Bisht et al. 2002b). As a constituent of 80s ribosomes, zinc deficiency leads to a loss of integrity of ribosomes. Meristematic tissue of zinc-deficient rice plants shows a decrease in the number of 80s ribosomes associated with a decrease in protein synthesis (Kitagishi et al. 1987). Role of zinc as a constituent of transcription factors may also contribute to decreased protein synthesis.

5.4,6, Auxin Metabolism

Visible symptoms such as stunting of shoot growth and epinastic curvature of leaves and decrease in auxin concentration of zinc-deficient plants (Skoog, 1940; Tsui, 1948) suggested involvement of zinc in auxin metabolism. Singh et al. (1981) found zinc-deficient rice plants to be low in tryptophan, which functions as the precursor of auxin in one of its biosynthetic pathways. Application of tryptophan to corn grown on Zn-deficient soil was also reported to benefit seedling growth (Salami and Kenfic, 1970). These observations were taken as suggestive of a role of zinc in tryptophan dependent of biosynthesis of IAA. Cakmak et al. (1989) reported Zn deprivation of bean plants to cause to stunting of shoot growth and leaf epinasty, associated with decreased endogenous level of IAA. Resupply of Zn to Zn-deficient plants restored the level of IAA to that found in the Zn-sufficient plants. Cakmak et al. (1989) attributed the Zn effect on shoot growth to oxidative degradation of IAA. Such a situation could results from Zn-deficiency induced activation of IAA decarboxylating peroxidases, commonly called IAA oxidase. Inhibitions of the shoot growth and leaf deformities form a common response of Zn deficiency, but they are not always associated with decrease in endogenous levels of IAA. In radish, Zn-deficiency produces symptoms characteristics of Zn-deficiency with little effect on IAA concentration in the shoots (Domingo et al. 1992; Hossain et al. 1998). Hossain et al. (1998) investigated effect of zinc on the alkali-labile fractions of IAA and found that their concentration in shoots of Zn-deficient and Zn-sufficient plants was about the same. They suggested the possibility of a role of Zn in influencing the biological activity of auxin.

5.4.7 Reproductive Development and Yield

Zinc nutrient status of plants plays an important role in plant reproduction. Its deficiency inhibits different stages of plant reproductive development. Effects range from induction/initiation of flowering, floral development, male and female gametogenesis, fertilization and seed development. Even under conditions of moderate zinc deficiency, when dry matter production is only marginally depressed, the development of anthers in wheat is severely retarded (Sharma et al. 1979b, 1987, 1990). When grown with inadequate zinc or when zinc supply is withheld at the onset of tassel emergence, maize plants show reduction in the size of anthers and poor development of sporogenous tissue. Sharma et al. (1987) showed that instead of forming sporogenous tissue (pollen mother cells), about 60% anthers of the top two florets of moderately Zn-deficient maize plants developed vascular tissue (vassels). The anthers of the Zn-deficient flowers displayed repression of male sexuality. Sharma et al. (1990) showed zinc to be critical fur microsporogenesis and pollen fertility. Inadequate supply of zinc leads to male sterility, impeding fertilization and seed setting.

Polar (1470) showed that pollen grains of broad bean contained a higher Concentration of zinc than in other plant parts. It was shown that the pollen tubes accumulated a larger proportion of zinc in the tip and that at least a part of it played a role in fertilization. Ender et al, (1983) showed higher accumulation of zinc in the growing tip of lily pollen than its basal parts. In field bean (Pandey et al. !995b) and green gram (Pandey et al. 2000), zinc deficiency has been shown to induce changes in exine morphology and reduce pollen viability. The exine of Zn-delident pollen grains lacks the reticulate and uniform muri present in the exine of the Zn-sufficient pollen grains. Instead, Zn-deficient pollen grains show highly sinuous, lobed muri having a waxy covering and raised baculae (Fig.5.2). Changes in exine pattern are reported to be caused by corresponding changes in mRNA and protein levels in pollen grains (Willing et al. 1988; Wetzel and Jensen, 1992), Zinc deficiency is reported to decrease the RNA content of pollen grains, possibly because of elevated levels of activity (Sharma et al. 197b; 1987). This may possibly lead to alterations in the exine pattern of Zn-deficient pollen grains. Zinc deficiency has also been shown to induce changes in stigma tic size, morphology and exudations,

Fig. 5.2 Scanning electron micrograph showing the effect of Zn deficiency on pollen m/c and exine architecture in green gram (V^fno radio ta (_ cv 1-44). The Zn-sufficient pollen grains (a) show reticulate ornamentation with uniform muri. In /.n-deficient pollen (fl)r reticulation is incomplete and muri are highly sinous and lobed. Muri also show w.m deposition and prominent raised baculae in the tumen (After Pandey and Sharma. 2000).

Fig. 5.2 Scanning electron micrograph showing the effect of Zn deficiency on pollen m/c and exine architecture in green gram (V^fno radio ta (_ cv 1-44). The Zn-sufficient pollen grains (a) show reticulate ornamentation with uniform muri. In /.n-deficient pollen (fl)r reticulation is incomplete and muri are highly sinous and lobed. Muri also show w.m deposition and prominent raised baculae in the tumen (After Pandey and Sharma. 2000).

inhibiting pollen-stigma interaction (Pandey et al. 1995b, 2005). Scanning electron microscopic (SHM) examination of the stigmatic surface of Zn-deficient field bean flowers revealed a decrease in the pollen receptive area of the stigmatic head.

Floral aberrations and inhibition of microsporogenesis in zinc-deficient plants may involve the role of zinc as a constituent of zinc finger proteins. The TF IIIA-type zinc linger proteins have been shown to control the development of floral parts by controlling cell division and/or expansion

Fig. 5.3. Scanning electron micrograph showing Jin-deficiency effect on stigma tic head of (aha bean iViiiu faba U and number of pollen adhering to it. Compared to 2n-sufficient stigma (A), only a few pollen grains are seen on the surface of Zn-deficiency stigma (B) which lacks in sligmatic exudations because of unruptured cuticle covering the papillae. (After Pandey et at. 1995).

Fig. 5.3. Scanning electron micrograph showing Jin-deficiency effect on stigma tic head of (aha bean iViiiu faba U and number of pollen adhering to it. Compared to 2n-sufficient stigma (A), only a few pollen grains are seen on the surface of Zn-deficiency stigma (B) which lacks in sligmatic exudations because of unruptured cuticle covering the papillae. (After Pandey et at. 1995).

of particular cell types in petunia (Takasuji et al. 1992) and Arabidopsis (Sakai et al. 1995). The anther-specific zinc finger proteins are suggested to play a role in microsporogenesis (Kobayashi et al. 1998) and the zinc finger polycomb proteins are suggested to be involved in megaga metagenesis and seed development (Grossrtikiaus et al. 1948; Brive et al. 2001). Reproductive development, including sporogenesis, could be impaired because of limited delivery of zinc to the reproductive organs. In a recent study, Takahashi et al. (2003) have shown severe suppression of reproductive development in transgenic tobacco plants (naas transgenics) lacking in synthesis of nicotianamine, which plays a role in the chelation of the cat ionic rnicronutrients and Iheir delivery to the reproductive tissues.

Seeds of zinc-deficient plants take a longer time to mature (Boawn el al. 1%9; Bay lock, 1995; Morghan and Grafton, 1999). Sharma et al, (1995b) reported alterations in seed coat topography, which could possibly inhibit their germination. Based on the study of Zn levels at different stages of seed development in Afabidopsis, Otegui et al. (2002) have reported accumulation of Zn, as Zn-phytate, in the chalazal vacuoles of the endosperm and its disappearance at the late globular stage. Expression of most seed proteins during embrvogenesis was taken to suggest that Zn-phytate stored in the chalazal vacuoles of the endosperm functions as the Zn source for the assembly of several diverse Zn-proteins, when their need arises during the course of embryo development (Otegui et al. 2002).

5.4.8 Water Relations

Suboptimal supply of zinc in sand culture induces changes in plant water relations parameters and leaf morphology, similar to those observed in response to water deficit. Sharma et al. (1982) showed that leaves of cabbage plants supplied with Zn-deficient nutrition turned thick and leathery and the leaf epidermis developed a thick waxy coating. The water content of the Zn-deficient leaves per unit leaf area was high, yet they showed higher water saturation deficit (WSD) and more negative water potential than corresponding leaves of Zn-sufficient plants. Similar observations were made for cauliflower. Limitation in zinc supply to cauliflower caused drastic reduction in stomatal opening, associated with increase in diffusive resistance and decrease in transpiration rate (Sharma and Sharma, 1987). Pandev and Sharma (2000) have described micro morphological changes in leaf epidermis of faba bean (Wan faba) grown at low (deficient) level of Zn supply (Fig. 5.4). The guard cells and the accessory cells showed concentric rings or folds (which became more prominent on removal of epicudcular wax); the stomatal lips were distorted and stomatal aperture was reduced (Sharma et al. 1995c; Pandev and Sharma, 2000).

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