Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

The plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry‐based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over‐producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

The glucosinolate breakdown product indole‐3‐carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana

The glucosinolate breakdown product indole‐3‐carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole‐3‐carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole‐3‐carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole‐3‐carbinol rapidly and reversibly inhibits root elongation in a dose‐dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole‐3‐carbinol and the auxin perception machinery was suggested, as application of indole‐3‐carbinol rescues auxin‐induced root phenotypes. In vitro and yeast‐based protein interaction studies showed that indole‐3‐carbinol perturbs the auxin‐dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3‐indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole‐3‐carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole‐3‐carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.     

New BAR tools for mining expression data and exploring Cis‐elements in Arabidopsis thaliana

Identifying sets of genes that are specifically expressed in certain tissues or in response to an environmental stimulus is useful for designing reporter constructs, generating gene expression markers, or for understanding gene regulatory networks. We have developed an easy‐to‐use online tool for defining a desired expression profile (a modification of our Expression Angler program), which can then be used to identify genes exhibiting patterns of expression that match this profile as closely as possible. Further, we have developed another online tool, Cistome, for predicting or exploring cis‐elements in the promoters of sets of co‐expressed genes identified by such a method, or by other methods. We present two use cases for these tools, which are freely available on the Bio‐Analytic Resource at http://BAR.utoronto.ca .     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin‐mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.     

Phytotyping4D: a light‐field imaging system for non‐invasive and accurate monitoring of spatio‐temporal plant growth

Integrative studies of plant growth require spatially and temporally resolved information from high‐throughput imaging systems. However, analysis and interpretation of conventional two‐dimensional images is complicated by the three‐dimensional nature of shoot architecture and by changes in leaf position over time, termed hyponasty. To solve this problem, Phytotyping^4D uses a light‐field camera that simultaneously provides a focus image and a depth image, which contains distance information about the object surface. Our automated pipeline segments the focus images, integrates depth information to reconstruct the three‐dimensional architecture, and analyses time series to provide information about the relative expansion rate, the timing of leaf appearance, hyponastic movement, and shape for individual leaves and the whole rosette. Phytotyping^4D was calibrated and validated using discs of known sizes, and plants tilted at various orientations. Information from this analysis was integrated into the pipeline to allow error assessment during routine operation. To illustrate the utility of Phytotyping^4D, we compare diurnal changes in Arabidopsis thaliana wild‐type Col‐0 and the starchless pgm mutant. Compared to Col‐0, pgm showed very low relative expansion rate in the second half of the night, a transiently increased relative expansion rate at the onset of light period, and smaller hyponastic movement including delayed movement after dusk, both at the level of the rosette and individual leaves. Our study introduces light‐field camera systems as a tool to accurately measure morphological and growth‐related features in plants.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

High‐resolution time‐resolved imaging of in vitro Arabidopsis rosette growth

Although quantitative characterization of growth phenotypes is of key importance for the understanding of essential networks driving plant growth, the majority of growth‐related genes are still being identified based on qualitative visual observations and/or single‐endpoint quantitative measurements. We developed an in vitro growth imaging system (IGIS) to perform time‐resolved analysis of rosette growth. In this system, Arabidopsis plants are grown in Petri dishes mounted on a rotating disk, and images of each plate are taken on an hourly basis. Automated image analysis was developed in order to obtain several growth‐related parameters, such as projected rosette area, rosette relative growth rate, compactness and stockiness, over time. To illustrate the use of the platform and the resulting data, we present the results for the growth response of Col–0 plants subjected to three mild stress conditions. Although the reduction in rosette area was relatively similar at 19 days after stratification, the time‐lapse analysis demonstrated that plants react differently to salt, osmotic and oxidative stress. The rosette area was altered at various time points during development, and leaf movement and shape parameters were also affected differently. We also used the IGIS to analyze in detail the growth behavior of mutants with enhanced leaf size. Analysis of several growth‐related parameters over time in these mutants revealed several specificities in growth behavior, underlining the high complexity of leaf growth coordination. These results demonstrate that time‐resolved imaging of in vitro rosette growth generates a better understanding of growth phenotypes than endpoint measurements.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

Inter‐regulation of the unfolded protein response and auxin signaling

The unfolded protein response (UPR) is a signaling network triggered by overload of protein‐folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down‐regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species‐specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER‐localized auxin transporters, including PIN5, we define a long‐neglected biological significance of ER‐based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone‐dependent strategy for coordinating ER function with physiological processes.     

Phenotiki: an open software and hardware platform for affordable and easy image‐based phenotyping of rosette‐shaped plants

Phenotyping is important to understand plant biology, but current solutions are costly, not versatile or are difficult to deploy. To solve this problem, we present Phenotiki, an affordable system for plant phenotyping that, relying on off‐the‐shelf parts, provides an easy to install and maintain platform, offering an out‐of‐box experience for a well‐established phenotyping need: imaging rosette‐shaped plants. The accompanying software (with available source code) processes data originating from our device seamlessly and automatically. Our software relies on machine learning to devise robust algorithms, and includes an automated leaf count obtained from 2D images without the need of depth (3D). Our affordable device (~€200) can be deployed in growth chambers or greenhouse to acquire optical 2D images of approximately up to 60 adult Arabidopsis rosettes concurrently. Data from the device are processed remotely on a workstation or via a cloud application (based on CyVerse). In this paper, we present a proof‐of‐concept validation experiment on top‐view images of 24 Arabidopsis plants in a combination of genotypes that has not been compared previously. Phenotypic analysis with respect to morphology, growth, color and leaf count has not been performed comprehensively before now. We confirm the findings of others on some of the extracted traits, showing that we can phenotype at reduced cost. We also perform extensive validations with external measurements and with higher fidelity equipment, and find no loss in statistical accuracy when we use the affordable setting that we propose. Device set‐up instructions and analysis software are publicly available ( http://phenotiki.com ).     

A toolkit for studying cellular reorganization during early embryogenesis in Arabidopsis thaliana

Considerable progress has been made in understanding the influence of physical and genetic factors on the patterns of cell division in various model systems. However, how each of these factors directs changes in subcellular structures has remained unclear. Generic machineries for the execution of cell expansion and division have been characterized, but how these are influenced by genetic regulators and physical cell properties remains an open question. To a large degree, the complexity of growing post‐embryonic tissues and a lack of precise predictability have prevented the extraction of rigid correlations between subcellular structures and future orientation of cell division. The Arabidopsis embryo offers an exquisitely predictable and simple model for studying such correlations, but so far the tools and methodology for studying subcellular structures in the early embryo have been lacking. Here, we describe a set of markers to visualize a range of subcellular structures in the early Arabidopsis embryo. We have designed a series of fluorescent cellular reporters optimized for embryos, and demonstrate the effectiveness of using these ‘ACE’ reporters with simple three‐dimensional imaging procedures that preserve delicate cellular structures. We describe the ontogeny of subcellular structures in the early embryo and find that central/peripheral cell polarity is established much earlier than suspected. In addition, we show that the actin and microtubule cytoskeleton has distinct topologies in the embryo. These tools and methods will allow detailed analysis of the events of cellular reorganization that underlie morphogenesis in the Arabidopsis embryo.     

Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell‐wall polysaccharides of Arabidopsis petals

A procedure for the simultaneous analysis of cell‐wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long‐chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell‐wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.     

Photoinducible DRONPA‐s: a new tool for investigating cell–cell connectivity

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell‐to‐cell transport. Here we report on an elegant non‐invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA‐s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA‐s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA‐s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA‐s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA‐s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA‐s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.     

Direct isolation of flavonoids from plants using ultra‐small anatase TiO2 nanoparticles

Surface functionalization of nanoparticles has become an important tool for in vivo delivery of bioactive agents to their target sites. Here we describe the reverse strategy, nanoharvesting, in which nanoparticles are used as a tool to isolate bioactive compounds from living cells. Anatase TiO₂ nanoparticles smaller than 20 nm form strong bonds with molecules bearing enediol and especially catechol groups. We show that these nanoparticles enter plant cells, conjugate enediol and catechol group‐rich flavonoids in situ, and exit plant cells as flavonoid‐nanoparticle conjugates. The source plant tissues remain viable after treatment. As predicted by the surface chemistry of anatase TiO₂ nanoparticles, quercetin‐based flavonoids were enriched amongst the nanoharvested flavonoid species. Nanoharvesting eliminates the use of organic solvents, allows spectral identification of the isolated compounds, and opens new avenues for use of nanomaterials for coupled isolation and testing of bioactive properties of plant‐synthesized compounds.