However, Al in the shoots exhibited no significant difference among rice varieties and the lowest contribution of varietal effect, likely due to the low phyto availability of Al colloidal precipitations in the slightly acidic hydroponic system, indicated that environmental factors also showed a marked influence on the rice ionomes. In contrast, the variance contributions of varietal effect to ionomic variations in Mg, Mn and Cu were even more than 60% in both shoots and roots. With large contributions of genotype effects in the shoots and roots, Mg, Mn and Cu were robust to environmental perturbations. In the present study, variations of Cr concentration in the shoots among all the subspecies were the largest, which was consistent with a previous study. Although the transport of Cr has been demonstrated to be associated with S transporters,such a significant difference among the rice genotypes indicated a complex underlying transport mechanism.The CVs of macro-nutrients excluding P in the roots in all rice subspecies were less than that of trace elements and heavy metals, indicating that the variations in macro-elements were stable among the rice varieties. Meanwhile, the lower CVs of nonessential elements in japonica demonstrating that micro-elements and heavy metal uptake in the roots showed greater genetic diversity in aus and indica than in japonica, which was consistent with previous reports that japonica subspecies exhibits less genetic and transcriptional diversity than indica and aus. The ionomic variation in rice varieties was mainly dependent on specific chemical element properties and genotype effects, as well as limitedly on subspecies. One of the most important values of ionomics is in determining the interactions between elements. The occurrence of antagonism and synergism between elements on uptake and translocation in plants has been reported in many studies. Correlation analysis showed that the rice subspecies and organs exhibited diverse strategies in establishing connections between elements, but many interactions were similar.
Ca was always significantly and positively correlated with Ba and Sr, and there was also positive correlation between Ba and Sr,vertical rack system indicating a significant positive correlation among divalent cations, possibly explained by their sharing the non-selective cation channel transporter-protein super families, such as ZIP, heavy metal ATPases and yellow stripe-like in the xylem due to their similar chemical properties and identical ionic valences. Significant positive correlations between P-As, P-Se and As-Se were detected in both shoots and roots among the subspecies. It has been reported that P and Se share the phosphate transporter OsPT2 gene. Moreover, Cao et al reported reduced As uptake in rice via a P transporter, OsPT4 gene knockout. In general, P application can activate the expression of OsPT2 and OsPT4 genes to improve As and Se uptakes in rice. These elemental interactions can provide a strategy to screen multi-element accumulation rice genotypes to breed rice varieties with abundant nutrients and that are safe to consume. Moreover, combined with the results of PCA on correlation coefficient, it was clear that the significantly positive correlations between minerals in the shoots of japonica were more obviously than those in indica and aus, while the patterns of ionomic correlations in the roots among the subspecies were similar. The elemental correlations in rice shoots mainly derive from ionomic transport, whereas in roots, it is due to element uptake, confirming that the ionomic differences were primarily caused by different transport mechanisms among the subspecies, consistent with previous studies. In rice domestication and breeding history, farmers have preferred to plant rice varieties adapted to the local agro-climatic conditions, with higher yield and better taste, usually not considering the micro-elements content,therefore the separation of elements in PCA mainly determined by different origins rather than subspecies. This finding further indicated that the genetic differences involving in ionomes in subspecies can vary with environmental changes. For example, the elemental differences in rice varieties from Japan mainly explained by most nonessential and toxic elements such as As and Cd, that can be related to the history of wide-ranging incidence of agricultural soil contamination in Japan.
Therefore, considering the geographical and historical distributions of rice varieties associated with the subspecies effects might be informative. Thus, due to the large genetic variation in different rice vareties, it is worthwhile to screen for rice varieties with higher nutrient concentrations, lower levels of toxic elements and healthier food values for use in bio-fortification strategies. The transportation of elements by the root-to-shoot process has been considered a rate-limitation factor in the shoot-tograin system. Consequently, identification of element accumulation in shoots is positively correlated with that in grains and determines the distribution of elements in grains. Additionally, owing to the numerous correlations between elements, the elemental covariation effects in shoots should be identified to determine the nutritional values and safety of rice varieties. In the present study, we identified many varieties accumulated a high concentration of multi-metallic elements. Higher concentrations of essential metal or metalloid elements in rice are important in bio-fortification to promote the synthesis of the coenzymes or proteins required for human health. Meanwhile, consistenting with the results of ionomic correlations in the shoots,multi-metal accumulation is likely involved in the non-selective metal cation channel transporter-protein super families. However, nonessential and toxic metals can be also indiscriminately accumulated in these varieties due to similar chemical properties, and potentially resulting in health risks to humans. JRC06 accumulated high concentrations of B, As, Co and Cr, while a lower concentration of K. As a macro-nutrient, K plays a highly significant role in alleviating abiotic stress in plants,explaining why metal uptake is likely suppressed, leading to a higher concentration of heavy metals. Both high K and Cs concentrations in the shoots of some rice varieties can be explained by the positive correlation between K and Cs,but it seemed contrary to previous studies. Ishikawa et al reported that the Cs concentration in rice is reduced by applying K, and Rai et al found that the expression of K transporter OsHAK1 in rice roots is the main route for Cs to accumulate in rice plants under a low K status. The plants in this study with a higher K concentration likely had a stronger affinity for Cs due to their similar chemical properties. However, the multi-element accumulation in almost all the aus and indica subspecies was lower than that in the japonica subspecies,indicating that the elemental covariations in the shoots showed subspecies differences and that the correlation of elements was stronger in japonica than in indica and aus, which was consistent with the results shown in Fig. 3.
The indica variety WRC11 showed high Zn and low Cd concentrations, while the aus variety WRC26 showed high Zn and low Cr concentrations. It has been reported that the OsHMA2 transporter in rice is associated with the co-transportation of Zn and Cd from roots to shoots, and that Zn competes with Cd by sharing the same ZIP transporter. Understanding and screening for rice varieties with significant correlations and potential for high nutritional value and safety are essential for rice breeding. In conclusion, this study revealed the ionomic responses to the genetic effects of rice varieties and subspecies under a strictly controlled environment. The ionomic differences among the subspecies were within the pre-framework of the genus Oryza, i.e., the concentrations of macro-nutrients were greater than micro-nutrients, and the micro-nutrients and anions were mainly restricted to the roots. The variations in the rice ionomes primarily depended on genetic factors and specific element chemical properties, and to a lesser extent, on subspecies factors and the geographical and historical distributions of the varieties. Moreover, the rice varieties screened for higher nutrient concentrations and beneficial elements in rice showed a higher value in variety breeding for human health. The potential health risks can be reduced by growing the varieties screened for low heavy metal accumulation even on heavy metalcontaminated soils. Therefore, the identification of rice varieties for bio-fortification and safe breeding should be closely related to the local edaphic conditions. Furthermore, the ionomic datasets of the rice genotypes in this study can provide a theoretical basis for transcriptional expression analyses of elementrelated proteins and transporters.Recirculating aquaponic systems,which grow both fish and plants, contain either a dedicated bio-filter and a hydroponic component for the plants, or a hydroponic component only that has the ability to act as a bio-filter. If balances between the biomass of fish grown and the nutrient removal ability of the plants are matched, the amounts of buffer required in aquaponic systems should be lower than that of fish-only systems. This is because plants are known to release negatively charged, alkalising ions when they are actively taking up nutrient ions such as nitrate. This is generally a one-to-one exchange mechanism since its primary function is to balance the homeostatic charge status within the plant root. However, the bacterial mediated nitrification of one ion of ammonia to one ion of nitrate releases two hydrogen ions ; therefore, the release of alkalising ions during active plant assimilation of nitrate cannot completely counteract the acidification caused by the nitrification of soluble fish waste ammonia. Therefore, as in fish-only RAS, mobile grow rack aquaponic systems often require the addition of a basic buffer, however due to the activity of the plants, this usually require less buffer additions than equivalent Fish-only RAS.
When buffer is added to a system,it is only the negative ion component that is used for pH buffering. Buffers are universally added as salts, as these are easier to acquire and handle. Hence, when buffers are added to aquatic systems, the positive ion component is unused and begins to accumulate within the system water. In fish-only systems, when buffered with sodium bicarbonate, sodium begins to accumulate. Sodium ion accumulation within fish-only systems is dealt with in the same manner as the build-up of other deleterious ions ; ionic concentrations are diluted by water exchange. One of the key advantages of recirculating aquaponic systems is that the addition of plants counteracts nutrient and other ionic accumulations, so that the reliance on water exchange to reduce ion accumulation is either lowered or negated completely. Therefore, aquaponic researchers have advocated the use of buffer salts to maintain pH that are based upon positive ions that are essential to plant growth and health, and therefore will not accumulate because the plants are actively using them in metabolic processes. Because fish feeds are generally lacking in potassium and calcium required for good plant growth, buffers based upon potassium and calcium salts are the most appropriate for aquaponic systems,as they provide the additional inputs of potassium and calcium that are essential for normal hydroponic plant metabolism. In previous experiments,sodium bicarbonate was used as the buffer species since it was the most common buffer used in fish-only, RAS culture. Whilst good plant growth was achieved using sodium bicarbonate as the buffer in this previous experiment, the current experiment was devised to test the suitability of both potassium-based and calcium-based buffers as alternatives, and to determine whether either of these buffers conferred advantages. The fish species used was the Australian native, freshwater fish Murray Cod, Maccullochella peelii, shown to be well adapted to aquaponic systems in previous experiments and the hydroponic vegetable was lettuce, Lactuca sativa. The system treatments were compared for fish growth, plant growth, buffer usage rate, nutrient accumulations,dissolved oxygen, pH, conductivity and water replacement rate.The experimental set-up was located, and all research was performed, within the Aquatic Culturing Laboratory Annex, Building 223, RMIT Bundoora Campus. The experimental, recirculating aquaponic system array consisted of 12 individual, identical aquaponic units, allowing replication of experimental treatments. Each aquaponic unit consisted of a fish holding tank, an associated bio-filter and a hydroponic component. The fish tank of each aquaponic unit was a round 100 L, opaque, white plastic tank. As well as fish, the tank contained an airlift pipe,a submersible water pump and a 100 W thermostatically controlled electrical resistance aquarium heater.