The calcium-dependent protein kinase CPK16 regulates hypoxia-induced ROS production by phosphorylating the NADPH oxidase RBOHD in Arabidopsis.

Reactive oxygen species (ROS) production is a key event in modulating plant responses to hypoxia and post-hypoxia reoxygenation. However, the molecular mechanism by which hypoxia-associated ROS homeostasis is controlled remains largely unknown. Here, we showed that the calcium-dependent protein kinase CPK16 regulates plant hypoxia tolerance by phosphorylating the plasma membrane-anchored NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to regulate ROS production in Arabidopsis (Arabidopsis thaliana). In response to hypoxia or reoxygenation, CPK16 was activated through phosphorylation of its Ser274 residue. The cpk16 knockout mutant displayed enhanced hypoxia tolerance, whereas CPK16-overexpressing (CPK16-OE) lines showed increased sensitivity to hypoxic stress. In agreement with these observations, hypoxia and reoxygenation both induced ROS accumulation in the rosettes of CPK16-OEs more strongly than in rosettes of the cpk16-1 mutant or the wild type. Moreover, CPK16 interacted with and phosphorylated the N terminus of RBOHD at four serine residues (Ser133, Ser148, Ser163, and Ser347) that were necessary for hypoxia- and reoxygenation-induced ROS accumulation. Furthermore, the hypoxia-tolerant phenotype of cpk16-1 was fully abolished in the cpk16 rbohd double mutant. Thus, we have uncovered a regulatory mechanism by which the CPK16-RBOHD module shapes ROS production during hypoxia and reoxygenation in Arabidopsis.

Nitrogen-mediated volatilisation of defensive metabolites in tomato confers resistance to herbivores.

Plants synthesise a vast array of volatile organic compounds (VOCs), which serve as chemical defence and communication agents in their interactions with insect herbivores. Although nitrogen (N) is a critical resource in the production of plant metabolites, its regulatory effects on defensive VOCs remain largely unknown. Here, we investigated the effect of N content in tomato (Solanum lycopersicum) on the tobacco cutworm (Spodoptera litura), a notorious agricultural pest, using biochemical and molecular experiments in combination with insect behavioural and performance analyses. We observed that on tomato leaves with different N contents, S. litura showed distinct feeding preference and growth and developmental performance. Particularly, metabolomics profiling revealed that limited N availability conferred resistance upon tomato plants to S. litura is likely associated with the biosynthesis and emission of the volatile metabolite α-humulene as a repellent. Moreover, exogenous application of α-humulene on tomato leaves elicited a significant repellent response against herbivores. Thus, our findings unravel the key factors involved in N-mediated plant defence against insect herbivores and pave the way for innovation of N management to improve the plant defence responses to facilitate pest control strategies within agroecosystems.

Transcriptome analysis reveals candidate genes involved in multiple heavy metal tolerance in hyperaccumulator Sedum alfredii.

Sedum alfredii Hance is a perennial herb native to China that can particularly be found in regions with abandoned Pb/Zn mines. It is a Cd/Zn hyperaccumulator that is highly tolerant to Pb, Cu, Ni, and Mn, showing potential for phytoremediation of soils contaminated with multiple heavy metals. A better understanding of how this species responds to different heavy metals would advance the phytoremediation efficiency. In this study, transcriptomic regulation of S. alfredii roots after Cd, Zn, Pb, and Cu exposure was analyzed to explore the candidate genes involved in multi-heavy metal tolerance. Although Zn and Cd, Pb and Cu had similar distribution patterns in S. alfredii, distinct expression patterns were exhibited among these four metal treatments, especially about half of the differentially expressed genes were upregulated under Cu treatment, suggesting that it utilizes distinctive and flexible strategies to cope with specific metal stress. Most unigenes regulated by Cu were enriched in catalytic activity, whereas the majority of unigenes regulated by Pb had unknown functions, implying that S. alfredii may have a unique strategy coping with Pb stress different from previous cognition. The unigenes that were co-regulated by multiple heavy metals exhibited functions of antioxidant substances, antioxidant enzymes, transporters, transcription factors, and cell wall components. These metal-induced responses at the transcriptional level in S. alfredii were highly consistent with those at the physiological level. Some of these genes have been confirmed to be related to heavy metal absorption and detoxification, and some were found to be related to heavy metal tolerance for the first time in this study, like Metacaspase-1 and EDR6. These results provide a theoretical basis for the use of genetic engineering technology to modify plants by enhancing multi-metal tolerance to promote phytoremediation efficiency.

Seasonal Zinc Storage and a Strategy for Its Use in Buds of Fruit Trees.

Bud dormancy allows deciduous perennial plants to rapidly grow following seasonal cold conditions. Although many studies have examined the hormonal regulation of bud growth, the role of nutrients remains unclear. Insufficient accumulation of the key micronutrient zinc (Zn) in dormant buds affects the vegetative and reproductive growth of perennial plants during the subsequent year, requiring the application of Zn fertilizers in orchard management to avoid growth defects in fruit trees. However, the mechanisms of seasonal Zn homeostasis in perennial plants remain poorly understood. Here, we provide new insights into Zn distribution and speciation within reproductive and vegetative buds of apple (Malus domestica) and four other deciduous fruit trees (peach [Amygdalus persica], grape [Vitis vinifera], pistachio [Pistacia vera], and blueberry [Vaccinium spp.]) using microscopic and spectroscopic characterization techniques comprising synchrotron-based x-ray fluorescence and x-ray absorption near-edge-structure analyses. By establishing a link between bud development and Zn distribution, we identified the following important steps of Zn storage and use in deciduous plants: Zn is preferentially deposited in the stem nodes subtending apical and axillary buds; Zn may then be sequestered as Zn-phytate prior to dormancy; in spring, Zn effectively releases for use during budbreak and subsequent meristematic growth. The mechanisms of Zn homeostasis during the seasonal cycles of plant growth and dormancy described here will contribute to improving orchard management, and to selection and breeding of deciduous perennial species.

Spatial imaging reveals the pathways of Zn transport and accumulation during reproductive growth stage in almond plants.

The reproductive processes of several deciduous trees are highly sensitive to Zn deficiency. An understanding of the patterns of Zn storage and remobilization during bud development and bud break is critical for the development of fertilization strategies to prevent deficiencies and may be valuable in selection and breeding programs to develop more Zn-resilient cultivars. In this study, we provide insights into the in situ distribution of Zn in almond reproductive organs at tissue, cellular, and subcellular scales using synchrotron-based X-ray fluorescence. The concentrations of Zn in different parts of the vegetative and reproductive tissues were also analysed. Our results show that the small branches subtending the flower and fruit, pollen grain, transmitting tissues of styles, and seed embryo are all important storage sites for Zn. An increase in Zn concentrations in almond reproductive organs mostly occur during the expanding growth phase, such as bud-flush and the mid-fruit enlargement stage; however, Zn transport to floral parts and fruit tissues was restricted at the pedicel and seed coat, suggesting a bottleneck in the export of Zn from the mother plant to filial tissues. Our results provide direct visual evidence for in-situ Zn distribution within the reproductive tissues of a deciduous tree species.

Efficient phloem remobilization of Zn protects apple trees during the early stages of Zn deficiency.

Apple trees are extensively cultivated worldwide but are often affected by zinc (Zn) deficiency. Limited knowledge regarding Zn remobilization within fruit crops has hampered the development of efficient strategies for providing adequate amounts of Zn. In the present study, Zn distribution and remobilization were compared among apple trees cultivated under different Zn conditions. Without Zn application, plants showed visible symptoms of Zn deficiency at the shoot tips after 1 year but appeared to grow normally during the first 6 months (early stage of Zn deficiency). Compared with apple plants under sufficient Zn treatment, plants suffering from early-stage Zn deficiency showed preferential Zn distribution to young leaves and higher Zn levels in phloem, demonstrating that hidden Zn deficiency triggers a highly efficient remobilization of Zn in this species. The in vivo Zn-nicotianamine complex in phloem tissues, combined with the significant enhanced expression of MdNAS3 and MdYSL6, suggested a positive role for nicotianamine in the phloem remobilization of Zn. These results strongly suggest that a proportion of Zn in the old leaves of apple trees can be efficiently remobilized by phloem transport to the shoot tips, partially in the form of Zn-nicotianamine, thus protecting apple trees against the early stages of Zn deficiency.

Cadmium Exposure-Sedum alfredii Planting Interactions Shape the Bacterial Community in the Hyperaccumulator Plant Rhizosphere.

Rhizospheric bacteria play important roles in plant tolerance and activation of heavy metals. Understanding the bacterial rhizobiome of hyperaccumulators may contribute to the development of optimized phytoextraction for metal-polluted soils. We used 16S rRNA gene amplicon sequencing to investigate the rhizospheric bacterial communities of the cadmium (Cd) hyperaccumulating ecotype (HE) Sedum alfredii in comparison to its nonhyperaccumulating ecotype (NHE). Both planting of two ecotypes of S. alfredii and elevated Cd levels significantly decreased bacterial alpha-diversity and altered bacterial community structure in soils. The HE rhizosphere harbored a unique bacterial community differing from those in its bulk soil and NHE counterparts. Several key taxa from Actinobacteria, Bacteroidetes, and TM7 were especially abundant in HE rhizospheres under high Cd stress. The actinobacterial genus Streptomyces was responsible for the majority of the divergence of bacterial community composition between the HE rhizosphere and other soil samples. In the HE rhizosphere, the abundance of Streptomyces was 3.31- to 16.45-fold higher than that in other samples under high Cd stress. These results suggested that both the presence of the hyperaccumulator S. alfredii and Cd exposure select for a specialized rhizosphere bacterial community during phytoextraction of Cd-contaminated soils and that key taxa, such as the species affiliated with the genus Streptomyces, may play an important role in metal hyperaccumulation.IMPORTANCESedum alfredii is a well-known Cd hyperaccumulator native to China. Its potential for extracting Cd relies not only on its powerful uptake, translocation, and tolerance for Cd but also on processes underground (especially rhizosphere microbes) that facilitate root uptake and tolerance of the metal. In this study, a high-throughput sequencing approach was applied to gain insight into the soil-plant-microbe interactions that may influence Cd accumulation in the hyperaccumulator S. alfredii Here, we report the investigation of rhizosphere bacterial communities of S. alfredii in phytoremediation of different levels of Cd contamination in soils. Moreover, some key taxa in its rhizosphere identified in the study, such as the species affiliated with genus Streptomyces, may shed new light on the involvement of bacteria in phytoextraction of contaminated soils and provide new materials for phytoremediation optimization.

Calcium Deficiency Triggers Phloem Remobilization of Cadmium in a Hyperaccumulating Species.

Understanding cadmium (Cd) accumulation in plants is critical for the development of plant-based strategies for soil remediation and crop safety. Sedum alfredii is a nonbrassica plant species known to hyperaccumulate Cd. The characteristics of Cd uptake, distribution, and retranslocation affected by the Ca status were investigated at cellular levels in S. alfredii Low Ca supply significantly increased Cd contents in shoots of S. alfredii, particularly in the young leaves. Micro x-ray fluorescence images confirmed that sequestration of Cd was greatly enhanced in the young leaves under Ca deficiency stress, with a significant amount of Cd localized in mesophyll cells, compared to the young leaves supplied with high Ca levels. Cd influx into protoplasts isolated from young leaves was significantly inhibited by the addition of Ca channel inhibitors, but not by pre-exposure to Ca deficiency. In stems, the Cd signal in vascular systems under low Ca levels was 10-fold higher than in those treated with higher Ca levels. A detailed investigation of vascular bundles revealed that an extremely high Cd signal induced by low Ca supply occurred in the phloem tissues, but not in the xylem tissues. Transfer of Cd pretreated plants to nutrient solutions at different Ca levels confirmed that a much higher amount of Cd was reallocated to the new growth tissues under low Ca stress compared to plants supplied with sufficient Ca. These results suggest that Ca deficiency triggered a highly efficient phloem remobilization of Cd in S. alfredii and subsequently enhanced Cd accumulation in its young leaves.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii.

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cd sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.

SaMT3 in Sedum alfredii drives Cd detoxification by chelation and ROS-scavenging via Cys residues.

Metallothioneins (MTs), a group of cysteine-rich proteins, are effective chelators of cadmium (Cd) and play a key role in plant Cd detoxification. However, little is known about the role of single cysteine (Cys) residues in the MTs involved in the adaptation of plants to Cd stress, especially, in hyperaccumulators. In the present study, we functionally characterised SaMT3 in S. alfredii, a Cd/Zn hyperaccumulator native to China. Our results showed that the C- and N- terminal regions of SaMT3 had differential functional natures in S. alfredii and determined its Cd hypertolerance and detoxification. Two CXC motifs within the C-terminal region were revealed to play a crucial role in Cd sensing and binding, whereas the four Cys-residues within the N-terminal region were involved in scavenging reactive oxygen species (ROS). An S. alfredii transgenic system based on callus transformation was developed to further investigate the in-planta gene function. The SaMT3-overexpressing transgenic plant roots were more tolerant to Cd than those of wild-type plants. Knockout of SaMT3 resulted in significantly decreased Cd concentrations and increased ROS levels after exposure to Cd stress. We demonstrated the SaMT3-mediated adaptation strategy in S. alfredii, which uses metal chelation and ROS scavenging in response to Cd stress. Our results further reveal the molecular mechanisms underlying Cd detoxification in hyperaccumulating plants, as well as the relation between Cys-related motifs and the metal binding properties of MTs. This research provides valuable insights into the functions of SaMT3 in S. alfredii, and improves our understanding of Cd hyperaccumulation in plants.

Rice cultivars significantly mitigate cadmium accumulation in grains and its bioaccessibility and toxicity in human HL-7702 cells.

Excessive Cd accumulation in cereals, especially in high-consumption staple crops, such as rice, is of major concern. Therefore, elucidation of cultivar-specific variation in rice grain Cd bioaccessibility and toxicity in humans would help the development of remedial strategies for Cd accumulation and toxicity. The present study combined an in vitro gastrointestinal digestion model with a human HL-7702 cell and assessed Cd bioaccessibility and toxicity to humans from the grains of 30 rice cultivars of different types harvested from Cd-contaminated paddy soil. The mean grain Cd content of cultivars within the type exceeded acceptable national standards. Cadmium bioaccessibility was high in all grains (9.08-23.6%) except the low accumulator (LA) rice cultivar (7.93%). The mean estimated daily intake of Cd via the cultivars (except LA) exceeded the FAO/WHO permissible limit based not only on the total grain Cd concentration but also on bioaccessible Cd concentration. A dose-proportional correlation between the in vitro bioaccessible and total grain Cd concentrations was observed, suggesting that Cd bioaccessibility accurately reflects the transfer of Cd from rice grain to humans. Toxicity assay results demonstrated that Cd from rice grains could commence oxidative stress and injury in HL-7702 cells, except the LA rice, which did not exhibit significant alteration in HL-7702 cells owing to its low Cd concentration. These results provide primary evidence to suggest that the cultivation of the LA rice cultivar is an effective agronomic approach to avert Cd entry into the food chain and alleviate Cd toxicity in humans.

Exogenous application of Mn significantly increased Cd accumulation in the Cd/Zn hyperaccumulator Sedum alfredii.

Sedum alfredii is a Cd/Zn hyperaccumulator native to China, which was collected from a mined area where Mn content in soil was extremely high, together with Zn and Cd content. We investigated the tolerance and accumulation ability of Mn and its possible association with Cd hyperaccumulation in this plant species by using MP-AES, SR-µ-XRF, and RT-PCR. The results showed that the hyperaccumulating ecotype (HE) S. alfredii exhibited high tolerance to Mn and accumulating around 10,000 and 12,000 mg kg-1 Mn in roots and shoots, respectively, without exhibiting toxicity under 5000 mg kg-1 Mn treatment for 4 weeks. Exposure to Cd significantly reduced plant uptake of Mn. In contrast, exogenous Mn application significantly improved root uptake and root-to-shoot translocation of Cd, resulting in the increased Cd accumulation in the shoots of HE S. alfredii. SR-µ-XRF analysis demonstrated that high Mn (20 µM) exposure resulted in higher intensities of Cd localized in both stem vascular bundles and cortex, as well as leaf mesophyll cells, than in those treated with low Mn levels (0.2 µM or 2.0 µM). RT-PCR analysis of several genes possibly involved in Mn/Cd transportation showed that expression of SaNramp3 in roots was significantly reduced under high Mn exposure. These results suggested a significant interaction between Cd and Mn in the HE S. alfredii plants, possibly through their competition for transporters and theoretically provided a strategy to improve the efficiency of Cd extraction from polluted soils by this plant species, after using appropriate nutrient management of Mn.

Penetration of foliar-applied Zn and its impact on apple plant nutrition status: in vivo evaluation by synchrotron-based X-ray fluorescence microscopy.

The absorption of foliar fertilizer is a complex process and is poorly understood. The ability to visualize and quantify the pathway that elements take following their application to leaf surfaces is critical for understanding the science and for practical applications of foliar fertilizers. By the use of synchrotron-based X-ray fluorescence to analyze the in vivo localization of elements, our study aimed to investigate the penetration of foliar-applied Zn absorbed by apple (Malus domestica Borkh.) leaves with different physiological surface properties, as well as the possible interactions between foliar Zn level and the mineral nutrient status of treated leaves. The results indicate that the absorption of foliar-applied Zn was largely dependent on plant leaf surface characteristics. High-resolution elemental maps revealed that the high binding capacity of the cell wall for Zn contributed to the observed limitation of Zn penetration across epidermal cells. Trichome density and stomatal aperture had opposite effects on Zn fertilizer penetration: a relatively high density of trichomes increased the hydrophobicity of leaves, whereas the presence of stomata facilitated foliar Zn penetration. Low levels of Zn promoted the accumulation of other mineral elements in treated leaves, and the complexation of Zn with phytic acid potentially occurred owing to exposure to high-Zn conditions. The present study provides direct visual evidence for the Zn penetration process across the leaf surface, which is important for the development of strategies for Zn biofortification in crop species.

Biochar significantly alters rhizobacterial communities and reduces Cd concentration in rice grains grown on Cd-contaminated soils.

Cadmium (Cd) contamination poses a serious problem in paddy soils. Biochar is frequently reported to deactivate Cd in soils and reduce Cd accumulation in rice plants, but few studies have addressed whether and how biochar affected the microbial communities in rice rhizosphere, which was an important factor determining the metal bioavailability and plant growth. In this study, biochar was pyrolyzed from bamboo (Phyllostachys heterocycla) chips at 350 °C. By using ICP-MS analysis and 16S rRNA gene sequencing, the impact of the biochar on Cd uptake by rice and on rhizospheric bacterial communities was investigated in both high-accumulating (HA) and low-accumulating (LA) rice cultivars grown in soils artificially contaminated with different Cd levels. Applied biochar significantly reduced Cd contents in rice plants of both cultivars, with substantially lower grain Cd contents for LA grown in highly contaminated soil. Soil pH was slightly increased by the applied biochar. Cd bioavailability was somehow reduced in soils, but not as significant as the reduction of Cd contents in rice plants. More interestingly, biochar application significantly altered the rhizobacterial community: it stimulated growth-promoting bacteria, such as Kaistobacter, Sphingobium (order Sphingomonadales), and Rhizobiaceae (order Rhizobiales); improved natural barrier formation and the transformation of metal mobilization around the rhizosphere mediated by, e.g., Rhodocyclaceae (class Betaproteobacteria) and Geobacter (class Deltaproteobacteria); and enhanced colonization of the LA rhizosphere possibly by taxa involved in Cd immobilization (Desulfovibrionales and Desulfobacterales). These results indicate that biochar application significantly reduces Cd uptake and accumulation by altering the rhizosphere bacterial community in rice grown on Cd-contaminated soils. The baseline data generated in this study provide insights that pave the way toward safer rice production.

Cultivar-specific response of bacterial community to cadmium contamination in the rhizosphere of rice (Oryza sativa L.).

Cadmium accumulation in rice grains is highly dependent on its bioavailability that affected by various physicochemical properties and microbiological processes of soil. The rhizospheric bacterial communities of rice grown in contaminated soils by means of rice cultivars highly or weakly accumulating Cd in grains (HA and LA, respectively) were investigated. HA roots absorbed 7.26- and 2.25-fold more Cd than did LA roots at low (0.44 mg kg-1) and high (6.66 mg kg-1) soil Cd levels, respectively. Regardless of Cd levels, Cd bioavailability in the rhizosphere of HA was significantly higher than that of LA. Planting of rice and elevated Cd levels both significantly decreased bacterial α-diversity and altered bacterial community structure, with noticeable differences between the rice cultivars. Taxa specifically enriched in the HA rhizosphere (phyla Bacteroidetes, Firmicutes, and Deltaproteobacteria) can directly or indirectly participate in metal activation, whereas the LA rhizosphere was highly colonized by plant growth-promoting taxa (phyla Alphaproteobacteria and Gammaproteobacteria). The results indicate a potential association of Cd uptake and accumulation with rhizosphere bacteria in rice grown on a contaminated soil, thus providing baseline data and a new perspective on the maintenance of rice security.

Spatial imaging and speciation of Cu in rice (Oryza sativa L.) roots using synchrotron-based X-ray microfluorescence and X-ray absorption spectroscopy.

Knowledge of elemental localization and speciation in rice (Oryza sativa L.) roots is crucial for elucidating the mechanisms of Cu accumulation so as to facilitate the development of strategies to inhibit Cu accumulation in rice grain grown in contaminated soils. Using synchrotron-based X-ray microfluorescence and X-ray absorption spectroscopy, we investigated the distribution patterns and speciation of Cu in rice roots treated with 50 µM Cu for 7 days. A clear preferential localization of Cu in the meristematic zone was observed in root tips as compared with the elongation zone. Investigation of Cu in the root cross sections revealed that the intensity of Cu in the vascular bundles was more than 10-fold higher than that in the other scanned sites (epidermis and cortex) in rice roots. The dominant chemical form of Cu (79.1%) in rice roots was similar to that in the Cu-cell wall compounds. These results suggest that although Cu can be easily transported into the vascular tissues in rice roots, most of the metal absorbed by plants is retained in the roots owing to its high binding to the cell wall compounds, thus preventing metal translocation to the aerial parts of the plants.

Supplemental macronutrients and microbial fermentation products improve the uptake and transport of foliar applied zinc in sunflower (Helianthus annuus L.) plants. Studies utilizing micro X-ray florescence.

Enhancing nutrient uptake and the subsequent elemental transport from the sites of application to sites of utilization is of great importance to the science and practical field application of foliar fertilizers. The aim of this study was to investigate the mobility of various foliar applied zinc (Zn) formulations in sunflower (Helianthus annuus L.) and to evaluate the effects of the addition of an organic biostimulant on phloem loading and elemental mobility. This was achieved by application of foliar formulations to the blade of sunflower (H. annuus L.) and high-resolution elemental imaging with micro X-ray fluorescence (µ-XRF) to visualize Zn within the vascular system of the leaf petiole. Although no significant increase of total Zn in petioles was determined by inductively-coupled plasma mass-spectrometer, µ-XRF elemental imaging showed a clear enrichment of Zn in the vascular tissues within the sunflower petioles treated with foliar fertilizers containing Zn. The concentration of Zn in the vascular of sunflower petioles was increased when Zn was applied with other microelements with EDTA (commercial product Kick-Off) as compared with an equimolar concentration of ZnSO4 alone. The addition of macronutrients N, P, K (commercial product CleanStart) to the Kick-Off Zn fertilizer, further increased vascular system Zn concentrations while the addition of the microbially derived organic biostimulant "GroZyme" resulted in a remarkable enhancement of Zn concentrations in the petiole vascular system. The study provides direct visualized evidence for phloem transport of foliar applied Zn out of sites of application in plants by using µ-XRF technique, and suggests that the formulation of the foliar applied Zn and the addition of the organic biostimulant GroZyme increases the mobility of Zn following its absorption by the leaf of sunflower.