In-situ phytoextraction of Ni by a native population of Alyssum murale on an ultramafic site (Albania)

Ultramafic outcrops are widespread in Albania and host several Ni hyperaccumulators (e.g., Alyssum murale Waldst. &; Kit.). A field experiment was conducted in Pojske (Eastern Albania), a large ultramafic area in which native A. murale was cultivated. The experiment consisted in testing the phytoextraction potential of already installed natural vegetation (including A. murale) on crop fields with or without suitable fertilisation. The area was divided into six 36-m² plots, three of which were fertilised in April 2005 with (NPK + S). The soil (Magnesic Hypereutric Vertisol) was fully described as well as the mineralogy of horizons and the localisation of Ni bearing phases (TEM-EDX and XRD). Ni availability was also characterised by Isotopic Exchange Kinetics (IEK). The flora was fully described on both fertilised and unfertilised plots and the plant composition (major and trace elements) and biomass (shoots) harvested individually were recorded. The soil had mainly two Ni-bearing phases: high-Mg smectite (1.3% Ni) and serpentine (0.7% Ni), the first one being the source of available Ni. Ni availability was extremely high according to IEK and confirmed by Ni contents in Trifolium nigriscens Viv. reaching 1,442 mg kg^?1 (A new hyperaccumulator?). Total biomass yields were 6.3 t ha^?1 in fertilised plots and 3.2 t ha^?1 in unfertilised plots with a highly significant effect: fertilisation increased dramatically the proportion of A. murale in the plots (2.6 t ha^?1 vs. 0.2 t ha^?1). Ni content in the shoots of A. murale reached 9,129 mg kg^?1 but metal concentration was not significantly affected by fertilisation. Phytoextracted Ni in total harvest reached 25 kg Ni ha^?1 on the fertilised plots. It was significantly lower in unfertilised plots (3 kg Ni ha^?1). Extensive phytomining on such sites could be promising in the Albanian context by domesticating already installed natural populations with fertilisation.     

Degradation of Alyssum murale biomass in soil

The Ni-hyperaccumulating plant Alyssum murale accumulates exceptionally high concentrations of nickel in its aboveground biomass. The reasons for hyperaccumulation remain unproven; however, it has been proposed that elemental alelopathy might be important. High-Ni leaves shed by the plant may create a "toxic zone" around the plant where germination or growth of competing plants is inhibited. The efficacy of this argument will partially depend upon the rate at which leaves degrade in soil and free metals are released, and the subsequent rate at which metals are bound to soil constituents. To test the degradation of biomass of hyperaccumulators, A. murale was grown on both high- and low-Ni soils to achieve high- (12.0 g Ni/kg) and low- (0.445 g Ni/kg) Ni biomass. Shredded leaf and stem biomass were added to a serpentine soil from Oregon that was originally used to grow high-Ni biomass and a low-Ni control soil from Maryland. Biomass Ni was readily soluble and extractable, suggesting near immediate release as biomass was added to soil Extractable nickel in soil amended with biomass declined rapidly over time due to Ni binding in soil These results suggest that Ni released from biomass of Ni hyperaccumulators may significantly affect their immediate niche only for short periods of time soon after leaf fall, but repeated application may create high Ni levels under and around hyperaccumulators.     

Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni(2+) toxicity

During the growth of Alyssum murale, a nickel accumulator plant, three root peptides chains of 55, 18 and 16kDa undergo phosphorylation. The intensity of the phosphorylated bands decreased in the course of growth in nutrient solution supplied with 0.5mM Ni(2+). In the shoot only two phosphorylated peptide chains with a size of 18 and 16kDa were detected. These two shoot peptides disappeared on the 19th day of growth in Ni(2+)-exposed plants, while the root peptide of 16kDa continued to be present in less intensity. This peptide was identified as the catalytic subunit of nucleoside diphosphate kinase (NDP kinase: E.C. 2.7.4.6) and was named NDPK-B. The enzyme was purified by means of ammonium sulphate precipitation, DEAE-sepharose and hydroxyapatite column chromatography. NDPK-B was thermostable, displayed a molecular mass of 103,000 and was comprised of six catalytic subunits. The autophosphorylated enzyme displayed an isoelectric point (pI) of 6.5. The NDPK-B autophosphorylation activity was metal-dependent. With regard to the transfer reaction, NDPK-B exhibited the following properties: (a) the enzyme had an optimum pH of 7.6; (b) it was capable of using both (gamma-(32)P) ATP and (gamma-(32)P) GTP as phosphate donors and of using all the available NDPs except dCDP as phosphate acceptors; (c) its activity using NDPs as substrates was metal dependent; (d) in the presence of (gamma-(32)P) GTP as the phosphate donor, it phosphorylated exclusively ADP when a mixture of NDPs was added in the reaction mixture; and, (e) ADP had a very low K(m) value towards 8.4nM. This high affinity towards ADP suggests that the enzyme may play a crucial function in the formation of the amount of ATP necessary for Alyssum murale to survive Ni(2+) stress.     

Cellular and subcellular compartmentation of Ni in the Eurasian serpentine plants Alyssum bracteatum, Alyssum murale (Brassicaceae) and Cleome heratensis (Capparaceae)

This study investigated the cellular and subcellular compartmentation of Ni in the Eurasian serpentine species Alyssum murale, Alyssum bracteatum and Cleome heratensis and a non-serpentine population of A. murale (as a control) grown in hydroponic culture. Plant growth responses and Ni uptake clearly revealed the higher Ni tolerance of serpentine plants than the non-serpentine plants. Serpentine A. murale and A. bracteatum grew better at elevated (0.01 mM) Ni in the nutrient solution, supporting the view that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Low shoot Ni content of C. heratensis in response to the high Ni treatments indicated that this species employs an avoidance strategy for Ni tolerance. Energy-dispersive X-ray microanalysis showed that Ni was highly concentrated in the cell walls and cell lumen, most likely the vacuoles, of leaf epidermis of A. murale and A. bracteatum rather than in the mesophyll cells. EDX spectra from leaves of the non-serpentine A. murale suggested that Ni accumulated in both epidermal and mesophyll cells but not in the epidermal cell walls. Growth reduction and Ni toxicity in plants of the non-serpentine A. murale could be due to accumulation of Ni in the lumen of leaf mesophyll cells. Our data suggest that cellular and subcellular compartmentation are both possible mechanisms for Ni tolerance employed by the serpentine A. murale and A. bracteatum.     

Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale

We have previously developed phytoremediation and phytomining technologies employing Alyssum Ni hyperaccumulators to quantitatively extract Ni from soils. Implementation of these technologies requires knowledge of Ni localization patterns for the Alyssum species/ecotypes of interest under realistic growth conditions. We investigated Ni uptake and localization in mature Alyssum murale Kotodesh and AJ9ç leaves. Seedlings were grown in potting mix with an increasing series of NiSO₄ addition (0, 5, 10, 20, 40, 80 mmol Ni kg^–1), NiC₄H₆O₄ addition (0, 5, 10, 30, 60, 90 mmol Ni kg^–1), in Ni-contaminated soil from metal refining operations, and serpentine soil. Plants at Ni levels 0, 5, 10, 20 mmolkg^–1 and in native soils grew normally. Plants at 40 mmolkg^–1 exhibited the onset of phytotoxicity, and 60, 80, and 90 mmolkg^–1 were demonstrably phytotoxic, but symptoms of phytotoxicity abated within 6 months. Cryogenic complement fractures were made from frozen hydrated samples. High-resolution scanning electron microscope (SEM) images were taken of one half. The other half was freeze-dried and examined with SEM and semi-quantitative energy dispersive x-ray analysis. Ni was highly concentrated in epidermal cell vacuoles and Ni and S counts showed a positive correlation. Trichome pedicles and the epidermal tissue from which the trichome grows were primary Ni compartments, but Ni was not distributed throughout trichomes. Palisade and spongy mesophyll and guard/substomatal cells contained lesser Ni concentrations but palisade mesophyll was an increasingly important compartment as Ni soil levels increased. Ni was virtually excluded from vascular tissue and trichome rays.     

Effects of nickel hyperaccumulation on physiological characteristics of Alyssum murale grown on metal contaminated waste amended soil

A pot experiment was conducted to investigate the effect of nickel concentration on physiological characteristics of Alyssum murale when grown in a soil mixed with sewage sludge (at the rate of 2.8%). Two types of sludge were used: agricultural sewage sludge (S1) and industrial sewage sludge with an increasing nickel concentration (S2, S3, and S4). Results showed that Ni in shoots was higher than Ni in roots. A. murale is able to concentrate up to 12730 mg/kg Ni in leaves. The highest dry matter yield was observed with plants grown with agricultural sewage sludge. An addition of S2 and S3 increased shoot biomass. However, application of S4 reduced 40% shoot dry weight as compared to the control Ni treatment did not affect all chlorophyll fluorescence parameters. The F(v)/F(m) ratio was stable between Ni treatments. Photosynthesis rate (A) increased with agricultural sewage sludge, but remained stable with variable Ni rates from the industrial sludge. The chlorophyll content increased with S1, S2 and S3 but it remains constant with S4 when compared to the control Therefore, high nickel concentration did not affect the function of the photosynthetic machine of A. murale.     

Interaction of nickel and manganese in accumulation and localization in leaves of the Ni hyperaccumulators Alyssum murale and Alyssum corsicum

The genus Alyssum contains >50 Ni hyperaccumulator species; many can achieve >2.5% Ni in dry leaf. In soils with normal Mn levels, Alyssum trichome bases were previously observed to accumulate Ni and Mn to high levels. Here we report concentration and localization patterns in A. murale and A. corsicum grown in soils with nonphytotoxic factorial additions of Ni and Mn salts. Four leaf type subsets based on size and age accumulated Ni and Mn similarly. The greatest Mn accumulation (10 times control) was observed in A. corsicum with 40 mmol Mn kg^?1 and 40 mmol Ni kg^?1 added to potting soil. Whole leaf Ni concentrations decreased as Mn increased. Synchrotron X-ray fluorescence mapping of whole fresh leaves showed localized in distinct high-concentration Mn spots associated with trichomes, Ni and Mn distributions were strongly spatially correlated. Standard X-ray fluorescence point analysis/mapping of cryofractured and freeze-dried samples found that Ni and Mn were co-located and strongly concentrated only in trichome bases and in cells adjacent to trichomes. Nickel concentration was also strongly spatially correlated with sulfur. Results indicate that maximum Ni phytoextraction by Alyssum may be reduced in soils with higher phytoavailable Mn, and suggest that Ni hyperaccumulation in Alyssum species may have developed from a Mn handling system.     

Improving the Agronomy of Alyssum murale for Extensive Phytomining: A Five-Year Field Study

Large ultramafic areas exist in Albania, which could be suitable for phytomining with native Alyssum murale. We undertook a five-year field experiment on an ultramafic Vertisol, aimed at optimizing a low-cost Ni-phytoextraction crop of A. murale which is adapted to the Balkans. The following aspects were studied on 18-m² plots in natural conditions: the effect of (i) plant phenology and element distribution, (ii) plant nutrition and fertilization, (iii) plant cover and weed control and (iv), planting technique (natural cover vs. sown crop). The optimal harvest time was set at the mid-flowering stage when Ni concentration and biomass yield were highest. The application of N, P, and K fertilizers, and especially a split 100-kg ha^?1 N application, increased the density of A. murale against all other species. It significantly increased shoot yield, without reducing Ni concentration. In natural stands, the control of graminaceous weeds required the use of an anti-monocots herbicide. However, after the optimization of fertilization and harvest time, weed control procured little benefit. Finally, cropping sown A. murale was more efficient than enhancing native stands and gave higher biomass and phytoextraction yields; biomass yields progressively improved from 0.3 to 9.0 t ha^?1 and phytoextracted Ni increased from 1.7 to 105 kg ha^?1.     

Potential for phytoextraction of137 Cs from a contaminated soil

Potential for phytoremediation of a soil contaminated with radiocesium was investigated in three phases: (1) hydroponic screening for plant species capable of accumulating elevated levels of cesium in shoots, (2) investigation of several amendments for their potential to increase the bioavailability of ¹³⁷Cs in the contaminated soil, and (3) bioaccumulation of radiocesium in shoots of plants grown in¹³⁷ Cs-contaminated soil.The bioaccumulation ratio for Cs in shoots of hydroponically grown plants ranged between 38 and 165. From solution, dicot species accumulated 2- to 4-fold more cesium in shoots than grasses. In studies investigating the bioavailability of ¹³⁷Cs in aged contaminated soil, ammonium salts were found to be the most effective desorbing agents, releasing approximately 25% of the¹³⁷ Cs. The extent of ¹³⁷Cs desorption from the soil increased with ammonium concentration up to 0.2 M. In a pot study conducted in a greenhouse, there was significant species-dependent variability in the ability to accumulate ¹³⁷Cs in the shoot from contaminated soil. The ability to accumulate ¹³⁷Cs from the soil increased in the order: reed canarygrass (Phalaris arundinacea) < Indian mustard (Brassica juncea) < tepary bean (Phaseolus acutifolius)< cabbage (B. oleracea var. capitata). It was also found that addition of NH₄NO₃ solution to the soil elicited a two- to twelve-fold increase in ¹³⁷Cs accumulation in the shoot. The greatest amount of ¹³⁷Cs (40 Bq g^-1 dw) was removed in shoots of cabbage grown in contaminated soil amended with 80 mmols NH₄NO₃ kg^-1 soil. Bioaccumulation ratios of 2–3 were obtained with the best performing plant species. These values are significantly greater than those previously reported in the literature (usually <0.1) for plants grown on aged contaminated soil. These results indicate that careful species selection along with amendments that increase the bioavailability of¹³⁷ Cs in the soil could greatly enhance the prospects for the use of plants to remediate ¹³⁷Cs-contaminated soils.     

Responses of two populations of an Iranian nickel-hyperaccumulating serpentine plant,Alyssum inflatum Nyar., to substrate Ca/Mg quotient and nickel

We selected two geographically close serpentine and non-serpentine populations of a Ni-hyperaccumulating plant (Alyssum inflatum) to investigate the influence of two common factors of serpentine soils: high Ni concentrations and low Ca/Mg quotients. Soils and plants were sampled from serpentine and non-serpentine substrates, and concentrations of Ca, Mg and Ni were measured. A hydroponic culture was used to compare growth and elemental composition responses of serpentine and non-serpentine plants to different Ca/Mg quotients and Ni concentrations in the nutrient solution. The Ca/Mg quotient for non-serpentine soils was 15 times higher than for serpentine soils, but there was no difference in the Ca/Mg quotient of plants from the two populations. In hydroponic culture, plants from both populations were able to survive at high Ca/Mg quotients. This result suggests that serpentine plants of A. inflatum do not necessarily need a substrate with a low Ca/Mg quotient for survival. Decreases in the Ca/Mg quotient in hydroponics decreased growth. The magnitude of this decrease was significantly greater in non-serpentine plants, suggesting a greater resistance of serpentine plants to low Ca/Mg quotients. Total Ni concentration in serpentine soils was 13 times higher than in non-serpentine soils, but ammonium nitrate-extractable concentrations of Ni in both soil types were similar. Ni concentrations in non-serpentine plants from their natural habitat were significantly lower than in serpentine plants, but there was no significant difference in Ni accumulation by plants of the two populations in hydroponic culture. However, increased concentrations of Ni in the hydroponic medium caused similar decreases in growth of both populations, indicating that Ni tolerance of the two populations was similar.     

Bacteria associated with yellow lupine grown on a metal‐contaminated soil: in vitro screening and in vivo evaluation for their potential to enhance Cd phytoextraction

In order to stimulate selection for plant‐associated bacteria with the potential to improve Cd phytoextraction, yellow lupine plants were grown on a metal‐contaminated field soil. It was hypothesised that growing these plants on this contaminated soil, which is a source of bacteria possessing different traits to cope with Cd, could enhance colonisation of lupine with potential plant‐associated bacteria that could then be inoculated in Cd‐exposed plants to reduce Cd phytotoxicity and enhance Cd uptake. All cultivable bacteria from rhizosphere, root and stem were isolated and genotypically and phenotypically characterised. Many of the rhizobacteria and root endophytes produce siderophores, organic acids, indole‐3‐acetic acid (IAA) and aminocyclopropane‐1‐carboxylate (ACC) deaminase, as well as being resistant to Cd and Zn. Most of the stem endophytes could produce organic acids (73.8%) and IAA (74.3%), however, only a minor fraction (up to 0.7%) were Cd or Zn resistant or could produce siderophores or ACC deaminase. A siderophore‐ and ACC deaminase‐producing, highly Cd‐resistant Rhizobium sp. from the rhizosphere, a siderophore‐, organic acid‐, IAA‐ and ACC deaminase‐producing highly Cd‐resistant Pseudomonas sp. colonising the roots, a highly Cd‐ and Zn‐resistant organic acid and IAA‐producing Clavibacter sp. present in the stem, and a consortium composed of these three strains were inoculated into non‐exposed and Cd‐exposed yellow lupine plants. Although all selected strains possessed promising in vitro characteristics to improve Cd phytoextraction, inoculation of none of the strains (i) reduced Cd phytotoxicity nor (ii) strongly affected plant Cd uptake. This work highlights that in vitro characterisation of bacteria is not sufficient to predict the in vivo behaviour of bacteria in interaction with their host plants.     

Biomass growth variation and phytoextraction potential of four Salix varieties grown in contaminated soil amended with lime and wood ash

Abstract

In a greenhouse experiment, plant growth and copper (Cu) and zinc (Zn) uptake by four Salix cultivars grown in Cu and Zn contaminated soils collected from a mining area in Finland were tested to assess their suitability for phytoextraction. The cultivars displayed tolerance to heavily contaminated soils throughout the experiment. After uptake, total mean Cu concentrations in the leaves, shoots and roots in all cultivars and treatments ranged from 163 to 474?mg kg^?1 and mean Zn concentrations ranged from 776 to 1823?mg kg^?1. Lime and wood ash addition increased dry biomass growth (25–43%), chlorophyll fluorescence (Fv/Fm) values (3–6%), the translocation factor (TF) (15–60% for Cu; 10–25% for Zn), the bio-concentration factor (BCF) (40–85% for Cu; 70–120% for Zn), and metal uptake (55–70% for Cu; 50–65% for Zn) compared to unamended treatment across all cultivars. The results revealed that Salix cultivars have the potential to take up and accumulate significant amounts of Cu and Zn. Cultivar Klara (Salix viminalis × S. schwerinii × S. dasyclados) was found to be the most effective cultivar for phytoextraction since it displayed greater dry biomass production, Fv/Fm, TF, BCF values and uptake percentage rates of Cu and Zn compared to the other three cultivars. This study indicates that further research is needed to clarify the wider phytoextraction capabilities of different Salix cultivars.     

Mechanical inhibition of hypocotyl elongation induces radial enlargement: implications for cytokinin action

Excised elongating segments from 3-day-old soybean (Glycine max var. Wayne) seedlings radially enlarged when auxin-promoted elongation was mechanically inhibited. Growth was similar to segments treated with auxin plus cytokinin. This observation suggests that cytokinin does not necessarily directly “reorient” cell enlargement. Cytokinin-induced radial cell growth may be a secondary effect of cytokinin's inhibition of auxin-promoted elongation.     

Biological diversity of Salix taxa in Cu,Pb and Zn phytoextraction from soil

The aim of the study was to estimate the efficiency of copper (Cu), lead (Pb) and zinc (Zn) phytoextraction by 145 Salix taxa cultivated in an area affected by industrial activity. Survivability and biomass of plants were also analyzed. The highest Cu, Pb and Zn content in shoots was 33.38 ± 2.91 (S. purpurea × viminalis 8), 24.64 ± 1.97 (S. fragilis 1) and 58.99 ± 4.30 (S. eriocephala 7) mg kg^?1 dry weight, respectively. In the case of unwashed leaves, the highest content of these metals was 135.06 ± 8.14 (S. purpurea 26), 67.98 ± 5.27 (S. purpurea 45) and 142.56 ± 12.69 (S. alba × triandra 2) mg kg^?1 dw, while in washed leaves it was 106.02 ± 11.12 (S. purpurea 45), 55.06 ± 5.75 (S. purpurea 45) and 122.87 ± 12.33 (S. alba × triandra 2) mg kg^?1 dw, respectively. The differences between the highest and lowest values for Cu, Pb and Zn were 545%, 20500% and 535% in shoots; 2692%, 2560% and 7500% in unwashed leaves; and 3286%, 2221% and 6950% in washed leaves, respectively. S. acutifolia was able to effectively accumulate all three metals jointly, producing shoots that were well developed in both length and diameter when compared with the other tested willows—an ability that would suggest its high suitability for practical application.     

Response of soil microbial activity to grazing, nitrogen deposition, and exotic cover in a serpentine grassland

Background and aims

Exotic species, nitrogen (N) deposition, and grazing are major drivers of change in grasslands. However little is known about the interactive effects of these factors on below-ground microbial communities.

Methods

We simulated realistic N deposition increases with low-level fertilization and manipulated grazing with fencing in a split-plot experiment in California’s largest serpentine grassland. We also monitored grazing intensity using camera traps and measured total available N to assess grazing and nutrient enrichment effects on microbial extracellular enzyme activity (EEA), microbial N mineralization, and respiration rates in soil.

Results

Continuous measures of grazing intensity and N availability showed that increased grazing and N were correlated with increased microbial activity and were stronger predictors than the categorical grazing and fertilization measures. Exotic cover was also generally correlated with increased microbial activity resulting from exotic-driven nutrient cycling alterations. Seasonal effects, on abiotic factors and plant phenology, were also an important factor in EEA with lower activity occurring at peak plant biomass.

Conclusions

In combination with previous studies from this serpentine grassland, our results suggest that grazing intensity and soil N availability may affect the soil microbial community indirectly via effects on exotic cover and associated changes in nutrient cycling while grazing directly impacts soil community function.     

Diversity and structure of ectomycorrhizal and co-associated fungal communities in a serpentine soil

The community of ectomycorrhizal (ECM) and co-associated fungi from a serpentine site forested with Pinus sylvestris and Quercus petraea was explored, to improve the understanding of ECM diversity in naturally metalliferous soils. ECM fungi were identified by a combination of morphotyping and direct sequencing of the nuclear ribosomal internal transcribed spacer region 2 and of a part of the large-subunit region. Co-associated fungi from selected ECM were identified by restriction fragment length polymorphism and sequencing of representative clones from libraries. Polymerase chain reaction with species-specific primers was applied to assess patterns of association of ECM and co-associated fungi. Twenty ECM species were differentiated. Aphyllophoralean fungi representing several basidiomycete orders and Russulaceae were dominant. Phialocephala fortinii was the most frequently encountered taxon from the diverse assemblage of ECM co-associated fungi. A ribotype representing a deeply branching ascomycete lineage known from ribosomal deoxyribonucleic acid sequences only was detected in some ECM samples. A broad taxonomic range of fungi have the potential to successfully colonise tree roots under the extreme edaphic conditions of serpentine soils. Distribution patterns of ECM-co-associated fungi hint at the importance of specific inter-fungal interactions, which are hypothesised to be a relevant factor for the maintenance of ECM diversity.     

Physiological adaptive mechanisms of plants grown in saline soil and implications for sustainable saline agriculture in coastal zone

There is large area of saline abandoned and low-yielding land distributed in coastal zone in the world. Soil salinity which inhibits plant growth and decreases crop yield is a serious and chronic problem for agricultural production. Improving plant salt tolerance is a feasible way to solve this problem. Plant physiological and biochemical responses under salinity stress become a hot issue at present, because it can provide insights into how plants may be modified to become more tolerant. It is generally known that the negative effects of soil salinity on plants are ascribed to ion toxicity, oxidative stress and osmotic stress, and great progress has been made in the study on molecular and physiological mechanisms of plant salinity tolerance in recent years. However, the present knowledge is not easily applied in the agronomy research under field environment. In this review, we simplified the physiological adaptive mechanisms in plants grown in saline soil and put forward a practical procedure for discerning physiological status and responses. In our opinion, this procedure consists of two steps. First, negative effects of salt stress are evaluated by the changes in biomass, crop yield and photosynthesis. Second, the underlying reasons are analyzed from osmotic regulation, antioxidant response and ion homeostasis. Photosynthesis is a good indicator of the harmful effects of saline soil on plants because of its close relation with crop yield and high sensitivity to environmental stress. Particularly, chlorophyll a fluorescence transient has been accepted as a reliable, sensitive and convenient tool in photosynthesis research in recent years, and it can facilitate and enrich photosynthetic research under field environment.     

Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis

Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4A connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a "plug-into-socket" binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.