The Physiological Characteristics Of Zinc Nutrition In High Zinc Density Rice (Oryza Sativa L.)

Zinc (Zn) is an essential micronutrient for all organisms, but its deficiency has become a widespread micronutrient malnutrition problem for human being. Rice (Oryza sativa L.) is a predominant staple food and a major source of dietary carbohydrate for more than half of the world's population; therefore, slight increase of Zn contents in rice grains can greatly improve human health. Increasing the Zn content in rice grains through breeding, which provided sufficient genetic variation of high Zn-density rice or through transgenic approaches, emerged as the times required and offered a suitable, cost-effective and sustainable approach to solve this problem. However, the physiological mechanism of zinc nutrition in high Zn density rice is not fully understood, which would limit the advance of high Zn density rice breeding. In this study, physiological mechanisms of Zn uptake, translocation and remobilization were investigated by using a stable isotope tracing method on two contrasting rice genotypes IR68144 (high Zn density) and IR64 (low Zn density) toghther with some factors influencing Zn accumulation in rice grains. The main results are summarized as followings:1. The Inductively Coupled Plasma Mass Spectrometry (ICP-MS) introduced in the early 1980s has the ability to determine isotope ratio rapidly. However, how to determine the content and ratio of traced element is not clear. By optimizing the operation parameters of the machine, solving interferes and studying the equipment stability, the determination method of 68Zn based on ICP-MS was set up which supplied an easy and trusty method to study zinc transportation, accumulation and metabolism in organism.2. Hydroponics experiments were carried out to compare Zn uptake and distribution in two contrasting Zn-density rice genotypes using stable isotope technique. A higher Zn concentration in xylem sap was observed compared to the uptake solution (even 20-fold higher), and it kept on increasing within 4-8 h while Zn concentration in the uptake solution was decreasing, indicating that root to shoot translocation of Zn in both rice genotypes is through symplastic passage. At seedling stage, high Zn density genotype IR68144 showed higher 68Zn uptake and transport rate to the shoot for the short-term (2 d), but no significant difference was observed in both genotypes for long-term (8 d). IR68144 exhibited higher Zn absorption ratio than IR64 at sufficient (2μM) or surplus (8μM) Zn supply level, and Zn in xylem sap of IR68144 was also consistently higher. However, IR64 and IR68144 showed similar patterns of 68Zn accumulation by new leaf at seedling stage and in developing grains at ripening stage, whereas 68Zn in new leaf and grains of IR68144 was consistently higher. These results suggested that a rapid root-to-shoot translocation and enhanced xylem loading capacity may be the crucial process for high Zn density in rice grains.3. The characteristics of Zn translocation and remobilization were investigated in high Zn density genotype IR68144, in comparison with the low Zn density genotype IR64. Stable isotope tracer 68Zn was supplied at various growth stages, either to the roots in nutrient solution or to the flag leaves to investigate the remobilization ability of 68Zn within phloem of rice plants and the foliar Zn effect of Zn accumulation in grains. The results showed that without external Zn supply at tillering stage, more 68Zn was retranslocated from "old tissues" (stem and old leaves) to "new tissues" (tillers and new growing leaves) in IR68144 than in IR64.71% of total Zn deposited in the fully expanded leaf of IR68144 was translocated to the new growing leaves and tillers, which was 63% in IR64. Despite of a higher 68Zn concentration in roots of IR64 and a higher translocation from root to shoot, a great portion of 68Zn remobilized was deposited in stem and thus could not be transported to new tissues (new growing leaves and tillers). Without Zn supply for 21 d,68Zn remobilized in IR68144 was 41.3% of total 68Zn in plants,1.2 fold higher of IR64. The accumulation of 68Zn in rice grains of IR68144 was also much higher than IR64 at reproduction stage, as much as 1.3 fold. Compared with the distribution rate of 68Zn in different tissues, we may figure out that 68Zn deposited in the developing grains of IR68144 was mainly transported from the stem, while from both of stem and root in IR64, which was consistent with the results of tillering stage experiment. Besides, foliar 68ZnSO4 could be translocated into rice grains through the phloem of flag leaves. The total Zn concentration of brown rice of treated plants was increased by 10% compared with CK, and a big portion of 68Zn absorbed by flag leaves could be translocated into grains. Retranslocation of 68Zn from flag leaves to grains was twice as high in IR68144 when 68Zn was applied to the flag leaves during booting or anthesis. These results indicate that Zn density in rice grains is closely associated with the ability to translocate Zn from old tissues to new tissues at both early and late growth stages and with phloem remobilization of Zn from leaves and stems to grains. 4. Stable isotope tracer 68Zn was supplied to the roots in nutrient solution at various growth stages to investigate the contribution of 68Zn absorbed at different growth stages to grain Zn accumulation. Significant differences in Zn allocation were observed between the two rice genotypes. Much higher Zn concentrations were found in grains, stems, and leaves of IR68144 than in IR64, which was 1.35,2.05,1.43 fold of IR64, respectively; but higher Zn was found in roots of IR64 as 2 fold of IR68144.68Zn absorbed by roots of both genotypes at various growth stages was mainly allocated in stem (32-62%), brown rice (10-33%), leaves (10-27%) and root (2-19%), very little was in hull (less than 2%). The entire rice growth period was divided into four stages:seedling (Ⅰ), tillering (Ⅱ), heading and anthesis (Ⅲ), and grain filling (IV). From calculation, we found that more than half of the Zn accumulated in the grains was remobilized before anthesis, accounting for 63% and 52% of the total Zn uptake for IR68144 and IR64, respectively. Only a little portion of Zn deposited in root and stem of both genotypes during stage II and stage III, most was transported into grains (brown rice and hull). Thus, irrespective of crops yield, these two stages could be the highest Zn utility efficiency stage. Besides, pot experiments showed that foliar ZnSO4 application increased the Zn concentration of shoots of rice plants grown in all of three soils, and Zn concentration in different tissues increased with increasing foliar ZnSO4 concentration. But Zn concentration increased was much higher in stem and leaves than in brown rice and hull, especially at the treatment of 1% ZnSO4). The content of other microelements (Fe, Mn, and Cu) and quality index varied with treatments and soil types. Mn concentration was not obviously affected by soil types and treatments, but Cu concentration was significantly affected by the soil types, which ranged that silty loam soil> purplish clayey soil> yellowish red soil and increased with foliar ZnSO4 concentration. Protein content in both genotypes was almost the same and not affected by foliar treatment. These results indicated that zinc allocated in the developing grains was mainly originated from the remobilized Zn, not from the part of Zn absorbed by root and directly transported in the xylem, and phloem remobilization of Zn from leaves and stems to grains could be the key factor for the high Zn density in rice grains. Foliar ZnSO4 application could significantly increase Zn concentration in rice grains though a lot of Zn was deposited in vegetative tissues, but this process might be affected by soil types.5. Hydroponics experiments were carried out to investigate the effects of three Zn sources (ZnSO4, Acetic Zinc and Zn-met) and three Zn levels treatments on the distribution of Zn in rice plants. The total Zn absorbed by the whole plant increased with Zn levels in both genotypes, but showed significant differences among different tissues. Under high Zn (4μM) treatment, a lot of Zn was allocated in stem and leaves, with an increase of 2-5 fold compared with CK (1μM), but only a little (by 30%-90%) increase was found in grains and thus leading to a decreased Zn distribution rate in grains with elevating Zn levels. At the same Zn level, more Zn could be absorbed by rice plants when applied as Acetic Zinc and Zn-met, as a significant higher grain Zn concentration was found than ZnSO4 under low Zn (0.25μM) level. Other microelements (Fe, Cu and Mn) in brown rice varied with different Zn sources and levels. Besides, milling characteristics showed that mass loss (y,%) correlated well with milling duration (x, s) and was fitted to the exponential equation y=aebx, so was Zn concentration (y, mg.kg"1) in grains. The relation index (R) was very close to 1, showed high relationship of Zn concentration and milling duration. When mass loss reached 10% (surface bran was removed), the loss Zn content was upto 30% of total Zn amount and Ca, P were much more than Zn, these results indicated that Zn (30%), Ca (50%) and P (65%) were stored in the grain surface of bran and embryo. The concentration of amylase and total protein was not changed with milling duration in both genotypes, but most of amino acids were decreased with milling duration, and were stable after 60s. These results indicated that increasing medium Zn concentration could improve the Zn absorption of rice plants to increase the concentration of Zn in rice grains, but too high Zn treatment results in big portion deposited in vegetative tissues and low Zn utility efficiency. Meanwhile, due to a non-uniform distribution of nutrients in the kernel, information on the distribution of nutrients in rice will greatly help in understanding the effect of milling and aid in designing procedures that improve technological and sensory properties of rice while retaining its essential nutrients as much as possible.