Proanthocyanidins (PAs), or condensed tannins, are widespread plant secondary metabolites common in trees. Proanthocyanidins play roles in plant defense and soil nutrient cycling, and have applications in human medicine and diet. Although PA function in plant shoots is well studied, there is less information on the role of PAs in roots. Proanthocyanidins can act as anti-fungal compounds, suggesting PAs in roots could negatively affect beneficial mycorrhizal fungi, and thus nutrient uptake. Poplars (Populus spp.) are known to produce a wide range of phenolic compounds, and for this work a transformable (P. tremula L. x P. tremuloides Michx.) hybrid was utilized. Transgenic lines with high and low tissue PA concentrations were used to test the hypothesis that high root PA levels would impede mycorrhizal colonization, and consequently, nitrogen uptake. Plants were grown in a sandwich tissue culture system allowing co-culture of the mycorrhizal fungi and roots. Plants from each line were inoculated with either the ectomycorrhizal (EcM) fungus Laccaria bicolor (Maire) P.D. Orton or the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler, or were kept as a non-inoculated control. Uptake of ammonium and nitrate by plant roots was measured by 15N-labeling. Successful EcM colonization on poplar roots was confirmed in all the plant lines, while no AM structures were observed. The low-PAs/phenolics line was less colonized by EcM fungi. When inoculated with EcM fungi, plants from all lines tended to have lower root PA concentrations. No significant differences in nitrogen uptake among plant lines were observed, but ammonium uptake was greater than nitrate uptake. Results suggest that PA content is reduced during colonization and that phenylpropanoids may play essential roles in establishing ectomycorrhizal symbioses.

Salicylic acid (SA) biosynthesis in plants occurs via the isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathways. The critical steps from benzyl-CoA to SA in the PAL-mediated pathway remain unknown. To probe benzenoid metabolism, we generated CRISPR/Cas9-mediated knockouts of benzaldehyde synthase in poplar. These plants produce less benzyl benzoate, benzyl salicylate and SA, yet accumulate more benzoic acid. We show that HSR203J encodes a carboxylesterase that specifically hydrolyzes benzyl salicylate. Virus-induced gene silencing (VIGS) of HSR203J in Nicotiana benthamiana led to reduced benzyl salicylate hydrolysis to SA. Based on these data, we propose a biosynthesis model and provide evidence that benzoyl-CoA is esterified to benzyl benzoate and converted to benzyl salicylate, which then releases SA. In addition, we identified a pathogen-induced cytochrome P450 encoded by HSR515 as a putative benzyl benzoate 2-hydroxylase. VIGS-mediated suppression of HSR515 in N. benthamiana reduced the conversion of benzyl benzoate to SA. Phylogenetic analyses indicated that Brassicaceae genomes do not contain HSR203J and HSR515 orthologs, whereas these genes are present in other vascular plants. These findings represent an important advance in our understanding of SA biosynthesis and identify missing steps in the PAL-mediated SA biosynthetic pathway.

Trees in the genus Populus synthesize sticky and fragrant resins to protect dormant leaf buds during winter. These resins contain diverse phenolic metabolites, in particular, hydroxycinnamate esters and methylated flavonoids. Populus trichocarpa leaf bud resin is characterized by methylated dihydrochalcone aglycones. To determine how the resin profile is influenced by seasonal changes, P. trichocarpa lateral leaf bud extracts and secreted surface resin were collected monthly over a 1-year cycle. The dihydrochalcones in both sets of extracts were quantified using ultrahigh pressure liquid chromatography-mass spectrometry (UPLC-MS), and other chemical changes were monitored using nontargeted metabolomics by UPLC-high-resolution MS (UPLC-HRMS). The results indicate that the dihydrochalcone content changes over the seasons and that biosynthesis occurs concomitant with bud development in the summer months. Nontargeted metabolomics data confirmed a pattern of dramatic changes in the summer and further suggested additional periods of substantive biochemical change in the resin. While overall patterns of surface-extracted resin matched those of whole bud extracts, some of the dynamics were shifted in the surface resin samples. This study provides the basis for the use of dihydrochalcones and other identified resin components as metabolic markers for more detailed investigations of resin biosynthesis, secretion, and movement to the bud surface.

Production of secreted leaf bud resin is a mechanism for temperate trees to protect dormant leaf buds against frost damage, dehydration, and insect herbivory. Bud resins contain a wide variety of special metabolites including terpenoids, benzenoids, and phenolics. The leaf bud resins of Populus trichocarpa and P. balsamifera contain high concentrations of O-methylated dihydrochalcones, but the enzymes for methylating these compounds remain enigmatic. We used transcriptomics and differential gene expression analyses to identify a gene encoding a P. trichocarpa dihydrochalcone-specific O-methyltransferase, which we named PtDOMT1. Detailed enzymatic analyses demonstrated PtDOMT1 to be a highly selective and regiospecific O-methyltransferase which methylates 4 and 4' positions of dihydrochalcones using S-adenosyl-l-methionine as a methyl donor. PtDOMT1 did not methylate any other flavonoid or phenolic substrate tested. These findings establish the final step in the biosynthesis of O-methylated dihydrochalcones in poplar and represent the first molecular analysis of leaf bud resin biosynthesis in plants.

Condensed tannins are common in vegetative tissues of woody plants, including in roots. In hybrid poplar (Populus tremula x alba; also known as P. x canescens) CT assays indicated they were most concentrated in younger white roots and at the root tip. Furthermore, CT-specific staining of embedded tissue sections demonstrated accumulation in root cap cells and adjacent epidermal cells, as well as a more sporadic presence in cortex cells. In older, brown roots as well as roots with secondary growth (cork zone), CT concentration was significantly lower. The insoluble fraction of CTs was greatest in the cork zone. To determine if CT accumulation correlates with nutrient uptake in poplar roots, a microelectrode ion flux measurement (MIFE™) system was used to measure flux along the root axis. Greatest NH4+ uptake was measured near the root tip, but NO3- and Ca2+ did not vary along the root length. In agreement with earlier work, providing poplars with ample nitrogen led to higher accumulation of CTs across root zones. To test the functional importance of CTs in roots directly, CT-modified transgenic plants could be important tools.

Proanthocyanidins (PAs) are common specialized metabolites and particularly abundant in trees and woody plants. In poplar (Populus spp.), PA biosynthesis is stress-induced and regulated by two previously studied transcription factors MYB115 and MYB134. To determine the relative contribution of these regulators to PA biosynthesis, we created single- and double-knockout (KO) mutants for both genes in transgenic poplars using CRISPR/Cas9. Knocking out either MYB134 or MYB115 showed reduced PA accumulation and downregulated flavonoid genes in leaves, but MYB134 disruption had the greatest impact and reduced PAs to 30% of controls. In roots, by contrast, only the MYB134/MYB115 double-KOs showed a significant change in PA concentration. The loss of PAs paralleled the lower expression of PA biosynthesis genes and concentrations of flavan-3-ol PA precursors catechin and epicatechin. Interestingly, salicinoids were also affected in double-KOs, with distinct patterns in roots and shoots. We conclude that the regulatory pathways for PA biosynthesis differ in poplar leaves and roots. The residual PA content in the double-KO plants indicates that other transcription factors must also be involved in control of the PA pathway.

Remobilization of carbon storage compounds in trees is crucial for the resilience to disturbances, stress, and the requirements of their perennial lifestyle, all of which can impact photosynthetic carbon gain. Trees contain abundant non-structural carbohydrates (NSC) in the form of starch and sugars for long term carbon storage, yet questions remain about the ability of trees to remobilize non-conventional carbon compounds under stress. Aspens, like other members of the genus Populus, have abundant specialized metabolites called salicinoid phenolic glycosides, which contain a core glucose moiety. In this study, we hypothesized that the glucose-containing salicinoids could be remobilized as an additional carbon source during severe carbon limitation. We made use of genetically modified hybrid aspen (Populus tremula x P. alba) with minimal salicinoid content and compared these to control plants with high salicinoid content during resprouting (suckering) in dark (carbon limited) conditions. As salicinoids are abundant anti-herbivore compounds, identification of such a secondary function for salicinoids may provide insight to the evolutionary pressures that drive their accumulation. Our results show that salicinoid biosynthesis is maintained during carbon limitation and suggests that salicinoids are not remobilized as a carbon source for regenerating shoot tissue. However, we found that salicinoid-producing aspens had reduced resprouting capacity per available root biomass when compared to salicinoid-deficient aspens. Therefore, our work shows that the constitutive salicinoid production in aspens can reduce the capacity for resprouting and survival in carbon limited conditions.

Climate and litter chemistry are major factors influencing litter decay, a process mediated by microbes, such as fungi, nitrogen-fixing bacteria and ammonia-oxidizing bacteria. Increasing atmospheric CO2 concentrations can decrease nitrogen (N) and increase condensed tannin (CT) content in foliar litter, reducing litter quality and slowing decomposition. We hypothesized that reduced litter quality inhibits microbes and is the mechanism causing decomposition to slow. Litterbags of Douglas-fir needles and poplar leaves with a range of N (0.61–1.57%) and CT (2.1–29.1%) treatment and natural acid unhydrolyzable residue (35.3–41.5%) concentrations were placed along climatic gradients in mature Douglas-fir stands of coastal British Columbia rainshadow forests. The structure (diversity, richness and evenness) and composition of microbial communities were analyzed using DGGE profiles of 18S, NifH-universal and AmoA PCR amplicons in foliar litter after 7, 12, 24 and 43 months of decay. High CT and low N concentrations in leaf litter were associated with changes in microbial community composition, especially fungi. Contrary to our hypothesis, high CT and low N treatments did not inhibit microbial colonization or diversity. The joint effects of air temperature and soil moisture on microbial community composition at our sites were more important than the effects of initial litter chemistry.

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Flavonoids, such as anthocyanins, proanthocyanidins, and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117, in hybrid poplar (Populus tremulaxtremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117-overexpressing poplars confirmed the up-regulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3',5'-hydroxylase (F3'5'H) genes. We also identified up-regulated cytochrome b(5) genes, required for full activity of F3'5'H . Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins, and flavonols in these transgenics. Similarly, overexpression of F3'5'H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of the cytochrome b(5) gene in F3'5'H1-overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by up-regulating F3'5'H1.

INTRODUCTION:The diarylheptanoid xyloside oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(β-d-xylopyranosyloxy)-heptan-3-one) has significant medicinal potential and is found at high concentration in leaves and bark of red alder (Alnus rubra).OBJECTIVES:To establish inexpensive and easily scaled methods for the extraction and purification of oregonin from timber by-products.METHODS:We developed a method combining aqueous extraction with spray drying of red alder extract into a powder, thus reducing the need for organic solvents used in traditional Soxhlet extraction or in solvent partitioning. Flash chromatography was utilised to purify oregonin from crude spray-dried alder extract.RESULTS:Crude spray-dried alder extract was comprised of an average of 9% of the diarylheptanoid compound oregonin. Less than 10% thermal degradation of oregonin was observed using extraction temperatures between 25°C and 50°C, followed by spray drying. The structure of purified oregonin was validated using high-performance liquid chromatography (HPLC), mass spectrometry (MS), ultraviolet spectroscopy (UV), and nuclear magnetic resonance (NMR).CONCLUSION:The developed method was robust, repeatable, and yielded purified oregonin of greater than > 95% purity (average of 95.8%). Our analysis represents the most complete NMR characterisation of oregonin reported to date.

Carwash wastewater (CWW) can be a significant source of environmental pollution due to the diversity and high concentrations of contaminants it contains. This toxic wastewater can contain several different heavy metals that if left untreated, can enter surface and sub-surface waters. Innovative, nature-based solutions such as low-impact development (LID) technologies may provide an eco-friendly CWW treatment process that is both effective and affordable. This research reviews the available literature to provide definitive values of flowrate and contaminant concentrations found in CWW around the globe. Dividing LID technologies into two groups, vegetated and unvegetated systems, the authors explored the literature for the general performance of these technologies to sustainably treat heavy metals in CWW. Depending on the car wash’s size and intended purpose, whether cleaning vehicles in agriculture-based rural communities, mining, or in high-density urban environments, volumetric flowrates requiring treatment found in six different countries ranged from 35–400 L/car. CWW also contains a wide range of contaminants at various levels, including COD, turbidity, TDS and TSS, surfactants, oils and greases, and heavy metals such as lead, cadmium, zinc, copper, chromium, and iron. Heavy metal removal by both vegetated and unvegetated LIDs shows mixed results in the literature, but given the different processes involved in both types, the authors propose a system that combines these types in order to provide all the necessary removal processes, including mechanical filtration, adsorption, sedimentation, chemical and biological treatment processes.

MYB transcription factors are central players in the regulation of phenylpropanoid and flavonoid metabolism. In proanthocyanidin and anthocyanin biosynthesis, MYBs act within a MBW (MYB-bHLH-WD40) complex. However, the MYB provides the specificity and ultimately determines where in the plant these pathways are activated. While work on the transcriptional regulation of proanthocyanidin biosynthesis in Arabidopsis seed coat has been instrumental in the discovery of MYB transcription factors, research in other plants such as grapevine and poplar has also made substantial contributions. In these systems, several types of MYB transcriptional activators of proanthocyanidin biosynthesis in poplar have been described, which in turn induce the expression of MYB repressors. The role of the MYB repressors may be to help modulate stress-induced PA synthesis, or to fine-tune flavonoid and phenylpropanoid metabolism. Characterizing both types of MYB genes via transgenic overexpression and promoter activation assays in transiently transformed cells has allowed us to construct a model for this gene regulatory network of MYB activators and repressors for regulation of proanthocyanidins in poplar. A long-term aim is to understand the mechanisms of repressor action, and to identify the linkages to upstream signals or environmental stimuli that induce proanthocyanidin accumulation.

Condensed tannins (CTs, proanthocyanidins) are widespread polymeric flavan-3-ols known for their ability to bind proteins. In poplar (Populus spp.), leaf condensed tannins are induced by both biotic and abiotic stresses, suggesting diverse biological functions. Here we demonstrate the ability of CTs to function as physiological antioxidants, preventing oxidative and cellular damage in response to drought and UV-B irradiation. Chlorophyll fluorescence was used to monitor photosystem II performance, and both hydrogen peroxide and malondialdehyde content was assayed as a measure of oxidative damage. Transgenic MYB-overexpressing poplar (Populus tremula × P. tremuloides) with high CT content showed reduced photosystem damage and lower hydrogen peroxide and malondialdehyde content after drought and UV-B stress. This antioxidant effect of CT was observed using two different poplar MYB CT regulators, in multiple independent lines and different genetic backgrounds. Additionally, low-CT MYB134-RNAi transgenic poplars showed enhanced susceptibility to drought-induced oxidative stress. UV-B radiation had different impacts than drought on chlorophyll fluorescence, but all high-CT poplar lines displayed reduced sensitivity to both stresses. Our data indicate that CTs are significant defences against oxidative stress. The broad distribution of CTs in forest systems that are exposed to diverse abiotic stresses suggests that these compounds have wider functional roles than previously realized.

Past work shows a significant negative correlation between foliar oregonin concentration and western tent caterpillar (Malacosoma californicum Packard) feeding on red alder (Alnus rubra Bong.). Above an oregonin threshold of 20% leaf dry weight, little feeding by caterpillars is observed. Concentrations of defensive chemicals are influenced by plant genotype, environmental conditions, insect feeding, and the interactions of these factors. Our objective was to measure the effects of nitrogen (N) availability and wounding on foliar oregonin and condensed tannin concentrations in red alder genotypes. One-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) for two growing seasons in a common garden. In the second year, leaves from 50 families from the fertilization experiment were used in a bioassay feeding experiment to determine the effects of N fertilization and genotype on WTC damage, and to identify a subset of 20 families with a range of damage to analyze for phytochemical composition. In separate experiments, wound-induction treatments were conducted outdoors and, in a greenhouse using the N treated trees in their third and fourth year, respectively. Foliar condensed tannin, oregonin and N concentrations were measured and ranked among the plant genotypes, and between the two N treatments and two wounding treatments. Results showed that oregonin and condensed tannin concentrations varied among the alder genotypes. Leaf N concentration was negatively correlated with concentration of oregonin. Neither of the measured phenolic compounds responded to wounding. The results suggest that red alder foliar oregonin and condensed tannin are likely constitutive defenses that are largely determined by genotype, and that the negative correlation of defense compounds with plant internal N status holds in this N-fixing tree.

Plants synthesize a wide range of bioactive secondary metabolites to defend against pests and pathogens. Red alder (Alnus rubra) bark, root, and leaf extract have a long history of use in traditional medicine and hygiene. Diarylheptanoids, especially oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(β-D-xylopyranosyloxy)-heptan-3-one), have been identified as major bioactive constituents. Diarylheptanoids have become a focus of research following reports of their antioxidant, antifungal, and anti-cancer activities. Recent data suggest that high oregonin concentration is associated with resistance of red alder leaves to western tent caterpillar (Malacosoma californicum) defoliation. Here we test effects of this compound directly on leaf-eating insects. Purified oregonin was examined in insect choice and toxicity tests using lepidopteran caterpillars. The compound exhibited significant anti-feedant activity against cabbage looper (Trichoplusia ni), white-marked tussock moth (Orgyia leucostigma), fall webworm (Hyphantria cunea), and M. californicum at concentrations corresponding to oregonin content of the most resistant alder clones in previous experiments. Toxicity tests were carried out with cabbage looper larvae only, but no contact or ingested toxicity was detected. Our results suggest that oregonin at levels found in red alder leaves early in the growing season may contribute to protecting red alder from leaf-eating insects.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

The salicinoids are anti-herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic, or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co-expression analysis with these two genes to identify two UDP-glucose dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicyl aldehyde, and 2-hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin, and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin, and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway.

The importance of the poplar MYB134 gene in controlling condensed tannin (CT) biosynthesis was tested by suppressing its expression using RNA interference (RNAi). MYB134-RNAi plants grew normally but showed reduced accumulation of stress-induced CTs in leaves. RNA-seq analysis indicated that flavonoid- and CT-related genes, as well as additional CT regulators, were strongly and specifically down-regulated by MYB134 suppression. This confirmed that the primary MYB134 target is the leaf flavonoid and CT pathway. Root CT accumulation was not impacted by MYB suppression, suggesting that additional CT regulators are active in roots and emphasizing the complexity of the regulation of CTs in poplar. To test the effect of CT down-regulation on oxidative stress resistance, leaves of MYB134-RNAi and control plants were exposed to the reactive oxygen species generator methyl viologen. MYB134-RNAi leaves sustained significantly more photosystem II damage, as seen in reduced chlorophyll fluorescence, compared with wild-type leaves. MYB134-RNAi leaves also contained more hydrogen peroxide, a reactive oxygen species, compared with the wild type. Our data thus corroborate the hypothesis that CT can act as an antioxidant in vivo and protect against oxidative stress. Overall, MYB134 was shown to be a central player in the regulation of CT synthesis in leaves.

This paper presents an eco-friendly, low-impact development (LID) approach, developed and implemented at a car wash site for a cleaner and greener environment. In this approach, the contaminated water discharged after car washing is treated as it is directed through an engineered, ecology-based water-management design. The design involves poplar (Populus deltoides) trees, Silva Cells®, and Fly-ash based Geopolymer concrete pavers working collectively to minimize the percolation of contaminants into the soil. In this novel system, each component has a vital role. For instance, the extensive roots of the poplar trees enable water filtration owing to phytoremediation effect; while the Silva Cells® promote stormwater management, planting of poplar trees and serve as a foundation for paver blocks. Lastly, the paver blocks made from industrial waste allow it to withstand urban load and infiltrate water runoff, thereby reducing runoff quantities. To evaluate the efficacy of contaminant uptake by this system, pH, electrical conductivity and turbidity, which are indicators of water quality levels, were monitored pre- and post-treatment. The percentage change in total dissolved solids indicates the potential of this treatment system for effective treatment of the contaminated car wash water.