OPT3 is a member of the oligopeptide transporter family and members of this family have been shown to mediate the transport of a broad range of peptides . Arabidopsis OPT3 has also been reported to rescue the ability of yeast mutants defective in Cu and Mn transport to grow on low concentrations of these transition metals . However, so far there is no direct evidence to suggest that OPT3 mediates the transport of transition metals, in the ionic form or complexed with a ligand, or whether OPT3 mediates the transport of a ligand that facilitates the uptake and accumulation of transition metals into the cell. In fact, our OPT3 localization experiments in yeast show that OPT3–YFP fusions are unable to transit out of the ER to the plasma membrane . This intracellular localization of OPT3 makes it difficult to interpret the ability of yeast strains defective in transition metal transport to grow on minimal media when expressing OPT3. Glutathione is a small peptide that has also gained recent attention in metal-status signaling via GSH coordinated intermediaries of the iron–sulfur cluster assembly machinery.Our radiotracer experiments using 35S-GSH and the ferric reductase assay in opt3-2 roots show that leaf-to-leaf movement of GSH was unaffected and that foliar application of GSH does not suppress the constitutive Fe-deficiency response in opt3-2. These results suggest that shoot-to-root transport of GSH alone has little effect on the long-distance signaling of the Fe status in Arabidopsis.Phloem transport plays a key role in delivering nutrients, including metals, hydroponic net pots to developing seeds . However, the mechanisms of toxic heavy metal loading into seeds are largely unknown. Nicotianamine, GSH, and PCs are the main metal-chelating molecules found in phloem sap . Nicotianamine has been shown to form complexes with Fe, Cu, Zn, and Mn, while GSH and PCs preferentially bind to Cd .
The differential partitioning of Cd among roots, leaves, and seeds in opt3-2 relative to the essential metals Fe, Zn, and Mn suggests that independent mechanisms mediate the partitioning of essential and non-essential metals, likely as specific metal–chelate complexes. Understanding phloem-mediated transport and seed loading mechanisms of individual metals and metal–ligand complexes will be important to restrict accumulation of toxic metals in seeds while ensuring the accumulation of essential metals. In summary, we show that Arabidopsis OPT3 is expressed in the phloem and functions in the long-distance shoot-to-root signaling of Fe/Zn/Mn status. When OPT3 expression is compromised, there is a mis-regulation of genes mediating uptake and mobilization of trace metals leading to an over-accumulation of cadmium, but not other metals, in seeds. We further show that mobilization of Fe2+ between leaves is impaired in opt3-2 and that targeted OPT3 expression in leaves is sufficient to restore Fe/Zn/Mn status signaling to roots providing molecular information on shoot-to-root Fe-status signaling. Sensing and regulation of trace-metal homeostasis in plants have been long-standing questions in plant biology and the results presented here offer new insights and avenues to advance our understanding of how essential and nonessential metals are accumulated and distributed within plant tissues.Wild-type and opt3-2 seeds were surface-sterilized, stratified at 4°C for 48h in the dark, and germinated under a 16-h light/8-h dark photoperiod. For Cd sensitivity experiments, ¼ MS plates were supplemented with 50 μM CdCl2 and allowed to grow vertically for 14 d. For metal determination in seeds, 2-week-old seedlings were transferred to Sunshine Basic Mix 2 soil supplemented with heavy metals as described . For metal determination in roots and leaves, plants were grown hydroponically as described previously . Elemental analyses were performed by ICP–OES at the UCSD/Scripps Institution of Oceanography analytical facility using dried rosette leaves, roots, or seeds digested overnight in trace-metal grade 70% HNO3 as described previously .In field experiments performed in co-operation with CIMMYT, 16 contrasting maize cultivars were tested for N efficiency and the underlying mechanisms for grain yield formation .
N-efficient cultivars were found to have a high N uptake and dry matter accumulation after anthesis, while N uptake and dry matter production until anthesis were not decisive for N efficiency. Further characteristics of N-efficient cultivars were delayed leaf senescence , a high harvest index and high kernel numbers . N uptake and dry matter production after anthesis have frequently been found to be decisive for grain yield in cultivar comparisons both under low and high N supply . In almost all these cases, cultivars with an improved performance during reproductive growth were also characterized by delayed leaf senescence. The causal relationships between the different traits are not yet clear. Genotypic differences in delayed leaf senescence might improve dry matter production after anthesis and thus increase harvest index and yield. It may also affect N uptake, due to an enhanced C supply to the roots. This view implies a key role for leaf senescence. However, delayed leaf senescence may also be merely a symptom of increased N uptake. To unravel the relationships between leaf senescence and the other traits decisive for N efficiency, correlation coefficients between the traits were calculated . Leaf senescence score 28 days after anthesis, with a high score representing a high ratio of senescent leaves on the plants, was negatively related to N efficiency in all investigated environments. Close relationships between leaf senescence score and dry matter accumulation and N uptake after anthesis, however, were found only in one of the experiments . This finding only partially support the above described assumption that delayed leaf senescence causes improved reproductive growth and N uptake. Surprisingly, leaf senescence score correlated with kernel numbers and harvest index suggesting that leaf senescence changes the pattern of N remobilization to the kernels. Thus, although delayed leaf senescence appears to be a decisive part of N efficiency and is suited as a selection trait for N efficiency, its physiological action remains to be elucidated. To test if leaf senescence might be a suitable selection trait for N efficiency also in short-term experiments, the same 16 tropical maize cultivars that were used for the field studies were grown in hydroponics . Leaf senescence was induced by subjecting the plants to N deficiency.
The progression of leaf senescence was monitored by photosynthesis and leaf chlorophyll measurements that were estimated by SPAD values. Cultivars differed both in SPAD values and photosynthesis rates of old leaves during N deprivation. Photosynthesis rate during leaf senescence proved to be a better indicator for N efficiency in this study than leaf chlorophyll content. Significant negative correlations were found between SPAD values, photosynthesis rates in the nutrient-solution experiment and leaf senescence scores in the field experiments, and positive correlations were found between photosynthesis rates and grain yield under low-N conditions in the field. The data suggests that the assessment of the capacity of a genotype to maintain a higher photosynthetic capacity of old leaves during N deficiency-induced senescence at the seedling stage may be suited as a selection parameter for N efficiency. However, photosynthesis rate during leaf senescence could explain only up to 20 % of the cultivar differences in N efficiency, while leaf senescence in the field experiments could explain 47 % . Enzymes within the chloroplast stroma are degraded early during leaf senescence which could be responsible for the decline in photosynthesis rate . Plant and leafN status at the beginning of the N deficiency period might influence the onset of leaf senescence. Plant-N status is determined by N uptake during early vegetative growth and depends on N supply during that period. An efficient root-N uptake rate during the N depletion period will prolong the N supply to the leaves. Apart from improving leaf-N status, this also increases cytokinin production of the roots , which will also delay leaf senescence . The leaf-senescence rate might also be influenced by the rate of N export from the leaf. The amount of N exported depends upon the breakdown of N compounds within the leaf and thus protease activity, but might also be influenced by sink strength. These findings raise the question if cultivar differences in N deficiency-induced leaf senescence might depend on the initial leaf-N content,blueberry grow pot which may be influenced by the N supply during leaf growth, the N uptake into the leaf or the total plant, or the amount of N which is exported from the leaf. Clarification of these aspects may help simplifying and/or improving the experimental procedure for an evaluation of N deficiency-induced leaf senescence in short-term experiments as a marker for N efficiency. Therefore, photosynthesis rates and leaf-N contents were investigated before and during N deficiency-induced leaf senescence for maize cultivars grown in hydroponics . The plants were pre-cultured at two different N rates thus creating differences in N status. Photosynthesis rates decreased considerably during leaf senescence; however, this was not always related to a decrease in leaf-N content of plants pre-cultured at low-N supply. In leaves of plants pre-cultured at high N supply, photosynthesis rates and leaf-N contents declined more in parallel. The decrease in photosynthesis rate must, therefore, have been governed by other factors than leaf-N status. This suggests that N remobilization was not the initial cause for N deficiency-induced leaf senescence, but may rather reflect leaf-inherent differences in leaf senescence. A dissection of N import and N export from the senescing leaf during the N-deficiency period was performed by 15N labelling.
Although there were only small net changes in leaf-N content during the N-deficiency period at N1, considerable N amounts were exported from and imported into the leaf during this time span . Leaf-N contents before the onset of leaf senescence were more than two times higher at high compared to low-N supply. The amount of N exported during N deprivation was nearly four times higher at high N compared to low N. These results suggest that N export was mainly governed by N availability in the leaf. Cultivar differences in leaf-N content prior to leaf senescence had no impact on leaf-N content during leaf senescence . Unexpectedly, N import represented a quantitatively not negligible part of total leaf-N even during leaf senescence, and cultivar differences in N import were also important for differences in total leaf-N during leaf senescence. Since N import was not related to total plant N uptake , it was probably governed by leaf-inherent factors. Some observations made by quantifying leaf and plant-N flows during N deficiency-induced leaf senescence were unexpected. First, photosynthesis rate decreased earlier and stronger than leaf-N content . This could be due to the degradation of N-containing enzymes within the chloroplast stroma . Alternatively, the declining photosynthesis rate induced leaf senescence and consequently N remobilisation from the leaf . Our results suggest that the decrease in photosynthesis rate might have been caused by a negative feedback regulation due to an accumulation of C assimilates in the leaves, since specific leaf weight increased during N deprivation . Leaf-area growth and thus shoot growth is strongly decreased by N deficiency mediated via cytokinins produced in the roots . A poor leaf growth will lead to a low carbohydrate demand and a low phloem-sap flow from matured leaves to growing leaves. This will also affect N retranslocation, since it could be shown that a low phloem-sap flow also decreases amino acid translocation . Thus, N retranslocation from senescing leaves under N deficiency might be delayed due to a low sink-N demand. N import might have played a decisive role for the induction of leaf senescence, since nitrate influx regulates the induction of leaf senescence . In this study, N import was probably governed by leaf-inherent factors instead of reflecting total plant-N uptake. Nitrate-N enters the leaf by the transpiration stream. Therefore, a decrease in stomatal conductance affects N import. This might be due to the decreased photosynthesis rate or mediated by abcisic acid , which is known to induce stomatal closure. Indeed, differences in ABA contents have been found in senescing leaves of an early-senescing and a stay-green phenotype of maize . The possible carbon and nitrogen flows in the plants which might influence leaf senescence of vegetative plants are summarized in Figure 2. Photosynthesis rates and leaf-N contents of plants pre-cultured at low or high N supply were significantly related to leaf senescence scores at anthesis of the same cultivars grown at low-N stress in the field .