Quantification analysis of H2O2 production Fig. 8B, measured as relative fluorescence as a percentage of control, showed that H2O2 levels were slightly, but not significantly, higher in the Cd-treated HE protoplasts compared with controls. In NHE protoplasts H2O2 formation occurred on a rapid timescale when under Cd stress and was significantly higher than that of controls at 30 min and 60 min .In hyper accumulators, the enhanced ability to protect roots from metal toxicity is facilitated partially through efficient shuttling of metals to the shoots . Our previous studies clearly showed a greatly enhanced rate of root-toshoot translocation was pivotal in the Cd hyper accumulator S. alfredii . The results in the present study confirm the highly enhanced transport of Cd in shoots of this plant species, as indicated by the marked enrichment of Cd within the vascular bundles of its stems and leaves after Cd exposure . The presence of metals within vascular systems is generally associated primarily with a xylem mode of transport and delivery of metals to aerial parts . The distribution patterns of Cd in stem tissues showed a larger proportion of Cd localized to the stem vascular bundles of HEs after treatments with 10 μM or 100 μM Cd for 7 d . This was not observed in NHEs . Based upon Cd signal intensity, the Cd content of stem vascular tissues of HEs was more than 10-fold higher than that of NHEs. This suggests that a considerable amount of Cd has been transported via the xylem vessels of HE S. alfredii,hydroponic grow kit probably as a result of its highly efficient xylem loading of Cd in its roots . The efficacy of Cd transport within xylem tissues and into shoots is further confirmed by the enrichment of Cd within the vascular tissues of young stems collected from HE S. alfredii plants treated with 100 μM Cd for 30 d . Tissue- and age-dependent variations in Cd distribution patterns in stems of HE S. alfredii were also identified in this study, similar to that previously reported for Zn in this plant species .

Vascular-enriched Cd was observed in stems treated with Cd for 7 d and young stems treated with Cd for 30 d, while Cd localization to the pith tissue and cortex layer was observed in the stems of HE S. alfredii exposed to Cd for 14 d or 30 d . This is consistent with previously reported results . Cd signal intensity in stem parenchyma cells of HE S. alfredii is more than 10-fold higher than those in vascular tissues of old stems after 30 d of Cd exposure, implying that sequestration of Cd is probably an active process in these tissues. This result is consistent with our previous work, which indicated that Cd is highly accumulated in the pith and cortex of stems and the mesophyll of leaves . The age- and tissue-dependent variation of Cd-enriched sites in stems implies efficient movement of Cd from vascular bundles into the pith and cortex during its translocation in shoots of HE S. alfredii. The parenchyma cells in these tissues, as well as leaf mesophyll , may serve specifically as terminal storage sites for Cd in this hyper accumulator plant species. This result is quite different from that reported for the other hyper accumulators, such as Noccaea Caerulescensand Arabidopsis halleri, where non-photosynthetic cells of the epidermis or trichomes accumulated the highest levels of Cd and there was a higher uptake rate of Cd into epidermal storage cells when compared with mesophyll cells . A possible explanation for Cd hyper accumulation in HE S. alfredii is that parenchyma cells in its shoot may have enhanced storage capacity for Cd sequestration. To evaluate the characteristics of Cd uptake by parenchyma cells in the shoots of HE S.alfredii, mesophyll protoplasts were isolated from the leaves of these plants and compared with those from NHE leaves. The results suggested metabolically dependent Cd uptake by mesophyll protoplasts of the two ecotypes, without significant differences in Cd accumulation. Transport of metal ions is generally an active process, which requires an energy supply as a driving force and selective binding sites . Furthermore, metabolically dependent uptake of metals by plants is essentially inhibited at low temperatures .

The significant inhibition of Cd uptake at low temperatures in both the time and concentration-dependent kinetics experiments suggests Cd uptake into the mesophyll protoplasts of both S.alfredii ecotypes is metabolically dependent. However, there was little difference in the time- and concentration dependent accumulation of Cd into mesophyll protoplasts between the two ecotypes . This suggests that the high capacity of Cd sequestration and tolerance in shoots of HE S. alfredii, at least for leaves, cannot be solely explained by its rapid cellular uptake rates. Similar results were reported for Cd uptake by leaf protoplasts of the hyper accumulators N. caerulescens and A. halleri , showing the absence of constitutively high transport capacities for Cd at the level of leaf protoplasts. A significant aspect of the differences between the two S. alfredii ecotypes is Cd transport into the vacuoles of mesophyll protoplasts. In most hyper accumulators, the metal is sequestered preferentially into compartments where they cannot impair metabolic processes . It makes sense for plants to store metals in their vacuoles since this organelle only contains enzymes such as phosphatases, lipases, and proteinases, which have not been identified as targets of heavy metal toxicity . It has been reported in the hyper accumulator N.caerulescens that Cd accumulated in the cytoplasm a few minutes after its addition and was then transported into vacuoles within its leaf cells . This is strongly supported by our observations of Cd distribution patterns in protoplasts of HE S. alfredii imaged using LeadmiumTM Green AM dye. Cadmium ion transport into vacuoles of protoplasts was not observed in NHEs, while Cd sequestration into vacuoles was consistently observed in protoplasts of HEs after 90–120 min exposure to Cd . This suggests that Cd ions were rapidly transported into vacuoles for storage, providing an efficient form of protection for the functional mesophyll cells in shoots of HE S. alfredii. This is supported by the results of viability and membrane integrity experiments , and by H2O2 imaging of Cd-treated mesophyll cells from the two ecotypes, which showed significantly higher Cd tolerance in HE mesophyll protoplasts than those of NHEs.

Here μ -XRF images of Cd in stems of HE S. alfredii , together with our previous studies , indicate that large amounts of Cd are stored in the pith and cortex cells after long-term Cd exposure. The essentially uniform distribution patterns of Cd in the pith and cortex cells of young stems shown by high resolution images further implied that considerable amounts of Cd are efficiently localized to the vacuoles of these parenchyma cells. It should be noted that freeze drying of the plant samples may have shifted dissolved Cd from the vacuole to the nearest available surface, hence localization of Cd in cell walls of the young stems in Fig. 2 is probably an artifact of sample preparation. As a succulent plant, S. alfredii has exceptionally large vacuoles in its parenchyma cells, making Cd storage there safer than it would be in regular sized mesophyll cells of other hyper accumulators . It is therefore logical that the efficacy of vacuolar sequestration by the parenchyma cells in shoots of HE S. alfredii is an important aspect of metal homeostasis and tolerance in this Cd hyper accumulator plant species. Taken together,hydroponic indoor growing system the results in the present study clearly demonstrate that one of the primary factors responsible for high Cd accumulation in HE S. alfredii is highly efficient root-to-shoot translocation, as suggested by the much enhanced Cd signal in the vascular bundles of its young stems. Furthermore, the efficient storage of Cd in vacuoles of the parenchyma cells, as opposed to its rapid transport into protoplasts, may represent a pivotal process in metal homeostasis and tolerance of cells in shoots of the Cd hyper accumulator HE S.alfredii. Combined with the idea that cellular uptake and sequestration of Cd are active processes within the terminal storage sites of HE S. alfredii, this study suggests that efficient transport across the tonoplast membranes within the parenchyma cells is the driving force for Cd hyper accumulation. This provides insights into specific translocation and storage strategies for Cd hyper accumulation in plants, particularly succulents that have large vacuoles in the thickened and fleshy leaf mesophyll.It is estimated that agriculture contributes 80% of anthropogenic nitrous oxide emissions , of which most are ultimately derived from nitrogen applied as N-fertilizers and manure-N . These agricultural N2O emissions account for about 5.5% of global annual anthropogenic GHG emissions . The N2O emitted originates from microbial N pathways, the balance of which is affected by the application of N fertilizers or by irrigation regime, aside from natural factors. Application of N fertilizers through micro-irrigation systems presents certain advantages over dry fertilization, especially in allowing more precise administration of N in coordination with crop demand . It has special relevance in arid and Mediterranean systems, where many high value tree crops are irrigated. Insufficient work has been done to describe the N2O emissions from N fertigation and to describe strategies of mitigation. Reports from fertigated tree crops have so far been lower than global expectations of N2O-N from mineral fertilizer applications, sometimes estimated as 0.9% of the total applied N , with a default Tier 1 value of 1% used by the IPCC . With various motives, renewed attention is being paid to “split applications” of nitrogen fertilizers, now usually called “high frequency” in the fertigation context. “High-frequency” N-fertigation in tree crops currently refers to at least 7 applications over the course of a growing season , and can reach up to daily applications during certain stages of development .

Monitored systems which adjust water and nutrient delivery to tree crops on an hourly basis are best described as “open hydroponics” . Increasing crop N-use efficiency may be the primary goal among growers who adopt high-frequency fertigation. Positive effects have been seen with daily application on low-OM, sandy soils with pomegranate , table grape and tomato . In Australia, high frequency N fertigation is becoming standard in citrus production, although yield differences in citrus have been elusive . Further crop-specific research should allow finer tuning for greater synchronicity with demand, as is sought by almond growers in California . Risks of nitrate leaching below the crop root zone accompany N fertigation, a problem with both productive and ecosystemic facets. Mediterranean trees such as almond can have strong fall root flushes with corresponding N uptake that aids against these losses. They can also have deep root systems, but they are not necessarily developed in such a way by farming practices, especially in drier climates . The utility of high-frequency fertigation in minimizing nitrate leaching is widely recognized particularly in sandy soils, or in soils with low cation exchange capacity . Physically, high-frequency reduces the peak soil solution N concentrations seen after fertigations and maintains root zones with more consistent levels of soluble nutrients . A larger fraction of applied N may be adsorbed on mineral or organic surfaces. And root physiological responses may increase N uptake efficiency. For similar reasons, high-frequency N-fertigation may also meet a third priority: reducing N2O emissions. At lower concentrations, N-processing soil microbes, whose rates are limited by a number of factors, are likely to transform a larger proportion of the applied N while the soil remains wet and conditions favorable. Among denitrifiers, with lower levels of available NO3, a lower fraction of this pool has been emitted as N2O , possibly in part because of inhibition of N2O reduction with greater NO3. The only study we are aware of that has compared N2O from high-frequency and standard Nfertigations was carried out under laboratory conditions , where it was found that splitting an application of KNO3 into 5 doses decreased total emissions. This result, too, would reflect the capacities of denitrifiers. Research at the field level is needed, since fertigation typically affects a large volume of soil, with relevant spatial variations in water-filled pore space, O2 and substrate concentrations. Field studies must also explore whether frequency of mineral N applications modifies soil microbial populations over time scales greater than the interval between applications, increasing the rates of certain transformations involved in nitrification and denitrification.