In Fe- and Zn-withholding treatments, root mineral content and concentration decreased substantially in both lines. Roots of all lines and treatments continued to grow during grain fill, and were larger at grain maturity than at anthesis, making calculation of remobilization by subtraction impractical. Despite the lack of significant net Fe and Zn remobilization from the total vegetative tissues of plants grown incomplete nutrient solution, the control line exhibited substantial Fe and Zn net remobilization from some vegetative organs . The control line exhibited a 54% and 48.3% decrease in flag leaf Fe and Zn content, respectively, between anthesis and maturity . For both minerals, the RNAi line remobilized significantly less Fe and Zn from the flag leaves. This indicates that the NAM genes influence net remobilization from flag leaves even in the complete nutrient solution. The other minerals tended to accumulate or remain constant in flag leaves between anthesis and 35 DAA, and the effect of the NAM genes was not readily apparent . An exception was Cu, which was also remobilized in both RNAi and control plants . These data suggest that under these experimental conditions,snap clamps ABS pvc pipe clip the effect of NAM genes on mineral remobilization primarily affects remobilizable metal micro-nutrients, i.e. Fe, Zn, and Cu.Over the time-course of the potting mix experiment, grain Ca and Mg concentrations were similar between lines at most time points, while K was higher in the RNAi line .
Copper, Fe, Mn, P, S, and Zn were at lower concentrations in the RNAi line at most time points. In the hydroponics experiments, the RNAi line produced more, although slightly smaller grains per head than the control line, resulting in significantly higher total grain mass per tiller . Across treatments, the total grain mineral content per tiller was higher in the RNAi line than in the control line for Ca and K, and similar for Mg, Mn, P, and S . This similar total grain mineral accumulation spread across a higher total grain weight in the RNAi line resulted in lower concentrations of Mg, Mn, P, and S in this line, relative to the control . Copper, Fe and Zn concentrations were also lower, but by a greater percentage than the other minerals. In terms of total grain accumulation , Fe was lower in the RNAi line relative to the control in complete solution at 35 DAA and 42 DAA , and in the Fe withheld experiment . Grain Zn content in the RNAi line was also significantly lower than the control in complete solution at 35 DAA and 42 DAA , but not in the Zn deficiency treatment .Much of the N imported into wheat grain is derived from protein in vegetative tissues that is degraded to amino acids and recycled by translocation to the grain, i.e. remobilization . It is often assumed that certain minerals supplied to seeds also come from remobilized sources, but, unlike N, specific sources of stored or recycled minerals are unknown, and the few studies that have quantified the contributions of minerals remobilized from vegetative tissues to seeds have reached differing conclusions .
In this work, net remobilization is defined as the loss of stored mineral content over time from one organ, and subsequent accumulation of that mineral content into another organ. Because net change in mineral content over time is a function of influx and efflux of nutrients, net remobilization will be detected only when efflux exceeds influx. Thus, substantial quantities of a given mineral could pass through an organ without a detectable change in content, resulting in no detectable net remobilization. Likewise, minerals could be remobilized from one subcellular compartment while accumulating in another compartment or in the apoplastic spaces without a change in total content or net remobilization. Since all shoot tissues have been collected and analysed in these experiments, it is possible to assess mineral partitioning to various tissues over time. If the grain mineral pool were to increase while the shoot mineral pool remained constant, then the quantity of mineral translocated to the grain must have passed through the shoot tissues, and would be equal to the quantity entering the shoot during that time period. When comparing lines, if translocation of mineral to the grain is inhibited, the decreased flux will be detected as a relative increase in vegetative mineral content and less of an increase in grain mineral content. Although this discussion will focus on Fe, Zn, and N, other minerals were quantified in order to determine whether the effects of the NAM genes were general in nature, or if certain minerals were disproportionately affected. Quantifying tissue DW and multiple minerals also demonstrated that remobilization did not occur for all minerals, and that observed changes in organ content were not secondary effects of changes in growth or organ mass.
The NAM genes are members of the NAC transcription factor family and were previously shown to affect grain Fe, Zn, and N content in a dosage-dependent manner. Construction of the RNAi line used here and the resultant alterations in NAM transcript levels have been described previously . As transcription factors, NAM proteins are predicted to regulate genes that encode for proteins that carry out physiological processes for nutrient remobilization and/or translocation to grain. The RNAi and control lines used in our work only differ in their relative NAM gene expression, and are otherwise isogenic. Therefore, differences in Fe, Zn, and N dynamics between the control and transgenic lines can be assigned to direct or downstream effects of these genes. Our results indicate that the extent of net remobilization is dependent on availability of mineral inputs and thus will probably be highly dependent on environmental conditions in field-grown plants. In complete hydroponic nutrient solution growth conditions, no significant net remobilization of Fe or Zn was observed in either line. Despite this, grain Fe and Zn contents and concentrations were substantially higher than those from plants grown in potting mix, where remobilization of both Fe and Zn was observed in control lines. When hydroponic plants were deprived of Fe or Zn inputs post-anthesis, net remobilization occurred in both the control and RNAi lines, from shoot tissues and also from roots . Both lines remobilized more than enough of these minerals to account for the entire grain content, although the Fe and Zn quantities accumulated in the grain were substantially lower than in plants on the complete solution treatment. These results suggest that while remobilization and partitioning of Fe and Zn to grain is impaired in the NAM knockdown line, this is not due to a complete inhibition of remobilization,greenhouse snap clamps as the RNAi line is capable of remobilizing minerals under nutrient-limiting conditions. They also suggest that when Fe and Zn are readily available to the roots, and are adequately absorbed into the plant, this source supersedes the need for remobilization from the leaves. In the absence of sufficient Fe and Zn from the soil, the plant can obtain these minerals from the storage forms present in both shoot and root vegetative tissues. These results are consistent with those obtained when P was withheld from wheat plants during grain development .In potting mix growth conditions, net remobilization of Fe and Zn from the control line was observed, but diminished or no net remobilization in the RNAi line. Total accumulation of plant Fe and Zn was similar, but partitioning of these minerals to grain was substantially lower in the RNAi line. In this same experiment, vegetative N content decreased in the control line over time, indicating net remobilization, while there was an increase in N in the RNAi line. Between 35 DAA and 56 DAA, net remobilized N could account for 46% of the increase in grain N in the control line, but accounted for none of the N in the grain of the RNAi line. These results, in combination with the 65Zn experiment that demonstrated decreased short-term translocation of Zn to grain, indicate that the translocation of certain minerals and N to grain is impaired in the NAM knockdown line.
A combination of decreased efflux and sustained influx of minerals into vegetative tissues could account for the lower net remobilization exhibited in the RNAi lines, and could also account for the lower percentage of total Fe, Zn, and N partitioned to grain. Target genes of the wheat NAM transcription factors have not been identified, and the molecular mechanism by which the NAM proteins affect translocation to grain is currently unknown. A micro-array study of senescence in Arabidopsis leaves revealed a large number of up-regulated transporter proteins, including OPTs, YSLs, and ZIPs . It is possible that NAM proteins regulate similar transporter genes in wheat and that these genes are needed for effective Fe and Zn remobilization. Other possible explanations include indirect effects on phloem loading for efflux of Fe, Zn and N from leaves; or an effect on the rate or timing of disassembly of the internal sources of these elements. The latter hypothesis is supported by the observation that lines with a functional copy of the NAM-B1 gene had higher soluble protein and amino acids concentration in the flag leaves than near isogenic lines with a non-functional NAM-B1 gene . The reduced expression of NAM genes and the accompanying delay of normal vegetative development, i.e. senescence, may result in a disruption of the normal source and sink tissue relationship. Delayed senescence and the accompanying degradation of proteins may result in a situation where the substrates for transporters are decreased or not present, or are only present later in the grain-filling period and thus are less efficiently translocated out of source tissues. Hundreds to thousands of proteins are estimated to interact with Zn ions as structural or catalytic components or as substrates , thus substantial quantities of Zn could be released during protein degradation. Similarly, Fe from the degradation of chloroplast proteins could be released during leaf senescence. Delayed degradation of chloroplasts containing these proteins, as suggested by the delay in leaf yellowing in the RNAi line, may explain why remobilization of Fe was inhibited proportionally more than the remobilization of Zn. Since the grain of the RNAi plants grew normally , the movement of water and photo assimilates did not seem to be impaired. This suggests an inhibition of translocation processes more specific to Fe, Zn, and N rather than a general inhibition of phloem transport.However, grain of control line wheat grown in complete hydroponic culture had a Zn concentration approximately five times higher , which parallels the improvements in Zn grain concentrations made via Zn fertilization . The grain Fe concentration of the hydroponic control line was approximately twice that of potting mix-grown plants . In the RNAi line also, Fe and Zn concentrations in grain from plants grown on complete hydroponic solution were significantly higher than in grain from potting mix-grown plants . These results suggest that wheat grain is already capable of accumulating several-fold higher Fe and Zn concentrations than are usually obtained in field situations. Because the RNAi line had lower partitioning of Fe and Zn to grain under both high and low availability, grain concentrations of these nutrients can possibly be increased by improvements in the efficiency of translocation. Indeed, over expression of an Arabidopsis Zn transporter in barley resulted in increased seed Zn concentration . However, constitutive over expression of a Zn transporter in rice resulted in the aberrant distribution of Zn within the plant . Over expression of transporters may need to be targeted spatially and temporally to result in the desired increases of nutrients in the target tissue. The transgenic line also showed reduced translocation of N. It is estimated that grain protein in the control line was 19.7%, while protein in the RNAi line was one-third lower, at 13.0%. These values are higher than normally observed in field situations, possibly as a result of the continuous supply of N to the plants. While decreased expression of the NAM genes negatively affects the accumulation of Fe, Zn and protein in the grains, increasing the transcript levels of the NAM genes above the levels normally found in current commercial varieties can result in increased protein.