Nicotianamine synthase 2 localizes to the vesicles of iron‐deficient rice roots,and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice

Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine (NA) synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2–sGFP fluorescence was observed in a dot‐like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di‐leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di‐leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2–sGFP plants were significantly higher than in non‐transformants and domain‐mutated lines, suggesting that OsNAS2–sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice.     

Uptake and transport of foliar applied zinc (65Zn) in bread and durum wheat cultivars differing in zinc efficiency

Using two bread wheat (Triticum aestivum) and two durum wheat (Triticum durum) cultivars differing in zinc (Zn) efficiency, uptake and translocation of foliar-applied ⁶⁵Zn were studied to characterize the role of Zn nutritional status of plants on the extent of phloem mobility of Zn and to determine the relationship between phloem mobility of Zn and Zn efficiency of the used wheat cultivars. Irrespective of leaf age and Zn nutritional status of plants, all cultivars showed similar Zn uptake rates with application of ⁶⁵ZnSO₄ to leaf strips in a short-term experiment. Also with supply of ⁶⁵ZnSO₄ by immersing the tip (3 cm) of the oldest leaf of intact plants, no differences in Zn uptake were observed among and within both wheat species. Further, Zn nutritional status did not affect total uptake of foliar applied Zn. However, Zn-deficient plants translocated more ⁶⁵Zn from the treated leaf to the roots and remainder parts of shoots. In Zn-deficient plants about 40% of the total absorbed ⁶⁵Zn was translocated from the treated leaf to the roots and remainder parts of shoots within 8 days while in Zn-sufficient plants the proportion of the translocated ⁶⁵Zn of the total absorbed ⁶⁵Zn was about 25%. Although differences in Zn efficiency existed between the cultivars did not affect the translocation and distribution of ⁶⁵Zn between roots and shoots. Bread wheats compared to durum wheats, tended to accumulate more ⁶⁵Zn in shoots and less ⁶⁵Zn in roots, particularly under Zn-deficient conditions. The results indicate that differences in expression of Zn efficiency between and within durum and bread wheats are not related to translocation or distribution of foliar-applied ⁶⁵Zn within plants. Differential compartementation of Zn at the cellular levels is discussed as a possible factor determining genotypic variation in Zn efficiency within wheat.     

Identification and localisation of the rice nicotianamine aminotransferase gene OsNAAT1 expression suggests the site of phytosiderophore synthesis in rice

Rice plants (Oryza sativa L.) take up iron using iron-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the biosynthetic pathway of MAs, nicotianamine aminotransferase (NAAT) catalyses the key step from nicotianamine to the 3′′-keto form. In the present study, we identified six rice NAAT genes (OsNAAT1–6) by screening a cDNA library made from Fe-deficient rice roots and by searching databases. Among the NAAT homologues, OsNAAT1 belongs to a subgroup containing barley functional NAAT (HvNAAT-A and HvNAAT-B) as well as a maize homologue cloned by cDNA library screening (ZmNAAT1). Northern blot and RT-PCR analysis showed that OsNAAT1, but not OsNAAT2–6, was strongly up-regulated by Fe deficiency, both in roots and shoots. The OsNAAT1 protein had NAAT enzyme activity in vitro, confirming that the OsNAAT1 gene encodes functional NAAT. Promoter–GUS analysis revealed that OsNAAT1 was expressed in companion and pericycle cells adjacent to the protoxylem of Fe-sufficient roots. In addition, expression was induced in all cells of Fe-deficient roots, with particularly strong GUS activity evident in the companion and pericycle cells. OsNAAT1 expression was also observed in the companion cells of Fe-sufficient shoots, and was clearly induced in all the cells of Fe-deficient leaves. These expression patterns highly resemble those of OsNAS1, OsNAS2 and OsDMAS1, the genes responsible for MAs biosynthesis for Fe acquisition. These findings strongly suggest that rice synthesises MAs in whole Fe-deficient roots to acquire Fe from the rhizosphere, and also in phloem cells to maintain metal homeostasis facilitated by MAs-mediated long-distance transport.     

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine (NA) and 2′‐deoxymugineic acid (DMA) – that play key roles in metal transport and nutrition. The CE‐OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE‐OsNAS2 grain and positively correlated with NA and DMA concentrations.     

The expression of iron homeostasis-related genes during rice germination

To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1–3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-β-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of chelator-buffered nutrient solution technique in studies on zinc nutrition in rice plant (Oryza sativa L.)

It has been difficult to impose different degrees of Zn deficiency on Poaceae species in nutrient solution because most chelators which would control Zn to low activities also bind Fe³⁺ so strongly that Poaceae species cannot obtain adequate Fe. Recently, a method has been developed to provide buffered Fe²⁺ at levels adequate for rice using Ferrozine (FZ), and use of other chelators to buffer the other micronutrient cations. The use of Fe²⁺ buffered with FZ in nutrient solutions in which Zn is buffered with HEDTA or DTPA was evaluated for study of Zn deficiency in rice compared to a conventional nutrient solution technique. The results showed that growth of rice plants in FZ+HEDTA-buffered nutrient solution was similar to that in the conventional nutrient solution. Severe zinc deficiency symptoms were observed in 28-day-old rice seedlings cultured with HEDTA-buffered nutrient solution at Zn²⁺ activities < 10^-10.6 M. With increasing free Zn²⁺ activities, concentrations of Zn, Fe, Cu, and Mn in shoots and roots were quite similar for the FZ+HEDTA-buffered nutrient solution and the conventional nutrient solution techniques. The percentages of water soluble Zn, Fe, Cu and Mn in shoots with HEDTA-buffered nutrient solution were also similar to those with the conventional solution. However, with DTPA-buffered nutrient solution, the rice seedlings suffered severe Fe deficiency; adding more FeFZ₃ corrected the Fe-chlorosis but shifted microelement buffering. Further, much higher total Zn concentrations are required to provide adequate Zn²⁺ in DTPA-buffered solutions, and the contents of Mn and Cu in shoots and roots cultured with DTPA-buffered solutions were much higher than those with the conventional or HEDTA-buffered solutions. In conclusion, DTPA-buffered nutrient solutions are not suitable but the FZ/HEDTA-buffered nutrient solution technique can be used to evaluate genotypic differences in zinc efficiency in rice.     

Mechanism of phosphorus-induced zinc deficiency in cotton. II. Evidence for impaired shoot control of phosphorus uptake and translocation under zinc deficiency

Cotton (Gossypium hirsutum L. cv. Deltapine 15/21) plants were precultured for 19 to 25 days under controlled climatic conditions in nutrient solutions with different levels of Zn. With the onset of visual Zn-deficiency symptoms the pH of the nutrient solution decreased from 6.0 to about 5.0. In contrast, Zn-sufficient plants raised the pH of the nutrient solution to about 7.0. In short-term studies it could be demonstrated that the Zn nutritional status of the plants remarkably influenced the uptake and translocation rates of mineral nutrients. Compared to Zn-sufficient plants, P uptake rate in severely Zn-deficient plants was increased by a factor of 2 to 3, whereas the uptake rates of K, Ca and particularly NO₃ decreased. The accumulation of P in the roots of Zn-deficient plants was either not affected or even lower than in Zn-sufficient plants. Thus, Zn deficiency had a specific enhancement effect on root to shoot transport of P. This enhancement effect of Zn deficiency on uptake and transport of P was similar at nutrient solution pH values of 7.0 and 5.8; i.e. it was not the result of acidification of the nutrient solution. After application of ³⁶CI, ⁸⁶Rb and ³²P to plant stems, basipetal transport of ³⁶CI and ⁸⁶Rb was not affected by the Zn nutritional status of the plants. However, in Zn-deficient plants, only 7.8% of the ³²P was translocated basipetally compared to 34% in the Zn-sufficient plants. A resupply of Zn for 19 h to Zn-deficient plants enhanced basipetal ³²P transport. The results indicate that a feedback mechanism in the shoots is impaired in Zn-deficient plants which controls the P uptake by roots and especially the P transport from roots to shoots. As a result of this impairment toxic concentrations of P accumulate in the leaves. The mechanism responsible is likely the retranslocation of P in the phloem from shoots to roots.     

Zinc uptake by rice as affected by iron and a chelator of ferrous iron

Summary Iron competitively inhibited Zn absorption by rice (Oryza sativa L. cv. Earlirose) grown in solution culture. The effect was more marked for shoots since Fe had also a competitive effect on Zn translocation from roots to shoots. The chelating agent baptholphenanthrolinesulfonate (BPDS), which has great ability to chelate Fe⁺⁺, alleviated the inhibitory effect of Fe to a large extent. re]19750516     

Root exudation and Fe uptake and transport in wheat genotypes differing in tolerance to Zn deficiency

Tolerance to Zn deficiency in wheat germplasm may be inversely related to uptake and transport of Fe to shoots. The present study examined eight bread (Triticum aestivum) and two durum (T. turgidum L. conv. durum) wheat genotypes for their capacity to take up and transport Fe when grown under either Fe or Zn deficiency. Bread wheat genotypes Aroona, Excalibur and Stilleto showed tolerance to Zn and Fe deficiency, while durum wheat genotypes are clearly less tolerant to either deficiency. Roots of bread wheats tolerant to Zn deficiency exuded more phytosiderophores than sensitive bread and durum genotypes. Greater amounts of phytosideophores were exuded by roots grown under Fe than Zn deficiency. A relatively poor relationship existed between phytosiderophore exudation or the Fe uptake rate and relative shoot growth under Fe deficiency. At advanced stages of Zn deficiency, genotypes tolerant to Zn deficiency (Aroona and Stilleto) had a greater rate of Fe uptake than other genotypes. Zinc deficiency depressed the rate of Fe transport to shoots in all genotypes in early stages, while advanced Zn deficiency had the opposite effect. Compared with Zn-sufficient plants, 17-day-old Zn-deficient plants of genotypes tolerant to Zn deficiency had a lower rate of Fe transport to shoots, while genotypes sensitive to Zn deficiency (Durati, Yallaroi) had the Fe transport rate increased by Zn deficiency. A proportion of total amount of Fe taken up that was transported to shoots increased with duration of either Fe or Zn deficiency. It is concluded that greater tolerance to Zn deficiency among wheat genotypes is associated with the increased exudation of phytosiderophores, an increased Fe uptake rate and decreased transport of Fe to shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alfalfa genotypes differ in their ability to tolerate zinc deficiency

Response of 13 alfalfa (Medicago sativa L.) genotypes to varied Zn supply (+Zn: 2 mg kg⁻¹ soil, −Zn: no added Zn) was studied in a pot experiment under controlled environmental conditions. Plants were grown for four weeks in a Zn-deficient siliceous sandy soil. Plants grown at no added Zn showed typical Zn deficiency symptoms i.e. interveinal chlorosis of leaves, yellowish-white necrotic lesions on leaf blades, necrosis of leaf margins, smaller leaves and a marked reduction in growth. There was solute leakage from the leaves of Zn-deficient plants, while no solute leakage from Zn-sufficient plants. The ratios of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn in Zn-deficient plants were extremely high compared with Zn-sufficient plants indicating disturbance of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn balance within plant system by Zn deficiency. Genotypes differed markedly in Zn efficiency based on shoot dry matter production. Alfalfa genotypes also differed markedly in P:Zn ratio, Cu:Zn ratio and Fe:Zn ratio under —Zn treatment. The shoot dry weight, shoot:root ratio, chlorophyll content of fresh leaf tissue, solute leakage from the leaves, Zn uptake and distribution of Zn in shoots and roots were the most sensitive parameters of Zn efficiency. Zn-efficient genotypes had less solute leakage but higher shoot:root ratio and higher Zn uptake compared with Zn-inefficient genotypes. Under —Zn treatment, Zn-inefficient genotypes had less Zn partitioning to shoots (33–37%) and more Zn retained in roots (63–67%), while Zn-efficient genotypes had about equal proportions of Zn in roots (50%) and shoots (50%). This revised version was published online in June 2006 with corrections to the Cover Date.     

Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions

Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants. In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis. Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants. Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA. Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts. In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots. We also isolated a genomic fragment containing both OsNAS1 and OsNAS2. In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves. The amounts of endogenous NA in both the roots and leaves were higher than in barley. We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation. Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes. Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.     

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background

Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g⁻¹ Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g⁻¹ Fe in endosperm.

Methodology/Principal Findings

Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g⁻¹ Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g⁻¹ Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions

The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.     

Zinc fertilisation increases tolerance to Rhizoctonia solani (AG 8) in Medicago truncatula

The effect of Zn fertilisation on tolerance of Medicago truncatula to infection by the root-rotting pathogen Rhizoctonia solani (AG 8) was studied in a field survey and in two experiments in controlled conditions. From the field survey, the concentration of Zn in the shoots of medics was found to be inversely related to the severity of disease on the root. Overall, the addition of Zn to Zn-deficient soil in controlled environment experiments resulted in reduced yield loss in the presence of R. solani, a reduction in disease score and no change in the concentration of nutrients in the shoots. However, under Zn deficiency, increasing levels of added R. solani resulted in significant yield loss, an increase in disease score and a reduction in concentration of Zn in the roots. This occurred despite a decrease in the number of infection sites caused by the fungus on the root and a lower amount of R. solani DNA extracted in medics deficient in Zn compared with plants supplied with Zn. While plants supplied with Zn were able to maintain a stable concentration of Zn in the shoots, the concentration of Zn in the roots also declined with increasing levels of R. solani. In conclusion, Zn application does not directly inhibit infection by R. solani, nor reduce its pathogenicity, but it does strongly increase root growth. The net result is that Zn-sufficient plants are more tolerant to the effects of root pruning by the fungus than Zn-deficient plants.     

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains

Background

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe + and Fe − medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe − conditions.

Results

HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe − condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna.

Conclusion

HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.     

Interspecies compatibility of NAS1 gene promoters.

Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying the regulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-acting elements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genes and understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous (Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiency induced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS induced strong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned in Arabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plants and Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On the other hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency. In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 and OsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compatible with some shoot-specific Fe deficiency-related trans-factors in tobacco.     

Effect of zinc fertilization on the mineral nutrition of rices differing in tolerance to zinc deficiency

Summary The effects of four Zn levels on the electrochemical and chemical properties of the soil solution, and on the growth and mineral nutrition of two rice varieties (IR26 and IR34) differing in tolerance to Zn deficiency were studied in the greenhouse using Zn-deficient soils from two locations. A similar experiment was conducted in culture solution to check how Zn addition affects translocation of other nutrients.In both soil and culture solution, plant Zn concentrations alone was not enough to account for varietal tolerance to Zn deficiency. Comparison of nutrient to Zn and shoot to root ratios of nutrients was more useful in determining the possible mechanism of varietal tolerance. IR 34 appeared to tolerate the disorder due to its lower Zn requirement, more efficient Zn translocation and ability to maintain lower Fe/Zn, Cu/Zn, Mg/Zn and P/Zn ratios in the shoot than the more susceptible variety, IR26. This was shown to be due to decreased translocation of Fe, Mg and P to shoots and decreased absorption of Cu by the root in IR34 in culture solution studies. Adding Zn further reduces translocation or absorption of these nutrients and depending on the nutrient supply of the soil, could cause deficiencies or mineral imbalances, especially of Fe, Cu, and P.These observed varietal differences regarding Zn requirement and the interaction of Zn with absorption and translocation of plant nutrients necessitates revision of recommendations for Zn fertilization. There is an inevitable need for Zn application in severely Zn-deficient soils regardless of rice variety. But on marginally Zn-deficient soils especially those low in Fe, Cu, or P, Zn fertilization is not advisable when resistant rice varieties are used.