Molecular bases for the stronger plastic response to high nitrate in the invasive plant Xanthium strumarium compared with its native congener.

MAIN CONCLUSION: High expressions of nitrate use and photosynthesis-related transcripts contribute to the stronger plasticity to high nitrate for the invader relative to its native congener, which may be driven by hormones. Strong phenotypic plasticity is often considered as one of the main mechanisms underlying exotic plant invasions. However, few studies have been conducted to investigate the related molecular mechanisms. Here, we determined the differences in the plastic responses to high nitrate between the invasive plant X. strumarium and its native congener, and the molecular bases by transcriptome analysis and quantitative real-time PCR validation. Our results showed that the invader had higher plasticity of growth, nitrogen accumulation and photosynthesis in responses to high nitrate than its native congener. Compared with its congener, more N utilization-related transcripts, including nitrate transporter 1/peptide transporter family 6.2 and nitrate reductase 1, were induced by high nitrate in the root of X. strumarium, improving its N utilization ability. More transcripts coding for photosynthetic antenna proteins were also induced by high nitrate in the shoot of X. strumarium, enhancing its photosynthesis. Hormones may be involved in the regulation of the plastic responses to high nitrate in the two species. Our study contributes to understanding the molecular mechanisms underlying the stronger plasticity of the invader in responses to high nitrate, and the potential function of plant hormones in these processes, providing bases for precise control of invasive plants using modern molecular techniques.

Amorphous mixed-valent Mn-containing nanozyme with cocklebur-like morphology for specific colorimetric detection of cancer cells via Velcro effects.

Designing nanozymes with excellent catalytic activity through valence state engineering and defect engineering is a widely applicable strategy. However, their development is hindered by the complexity of the design strategies. In this work, we employed a simple calcination method to regulate the valence of manganese and crystalline states in manganese oxide nanozymes. The oxidase-like activity of the nanozymes was found to benefit from a mixed valence state dominated by Mn (III). And the amorphous structure with more active defect sites significantly enhanced the catalytic efficiency. Moreover, we demonstrated that amorphous mixed-valent Mn-containing (amvMn) nanozymes with unique cocklebur-like biomimetic morphology achieved specific binding to cancer cells through the Velcro effects. Subsequently, the nanozymes mediated TMB coloration through their oxidase-like activity, enabling the colorimetric detection of cancer cells. This work not only provides guidance for optimizing nanozyme performance, but also inspire the development of equipment-free visual detection methods for cancer cells.

DELAY OF GERMINATION 1 (DOG1) regulates dormancy in dimorphic seeds of Xanthium strumarium.

Seed dormancy ensures plant survival but many mechanisms remain unclear. A high-throughput RNA-seq analysis investigated the mechanisms involved in the establishment of dormancy in dimorphic seeds of Xanthium strumarium (L.) developing in one single burr. Results showed that DOG1 , the main dormancy gene in Arabidopsis thaliana L., was over-represented in the dormant seed leading to the formation of two seeds with different cell wall properties. Less expression of DME /EMB1649 , UBP26 , EMF2, MOM, SNL2, and AGO4 in the non-dormant seed was observed, which function in the chromatin remodelling of dormancy-associated genes through DNA methylation. However, higher levels of ATXR7 /SDG25, ELF6 , and JMJ16/PKDM7D in the non-dormant seed that act at the level of histone demethylation and activate germination were found. Dramatically lower expression in the splicing factors SUA, PWI , and FY in non-dormant seed may indicate that variation in RNA splicing for ABA sensitivity and transcriptional elongation control of DOG1 is of importance for inducing seed dormancy. Seed size and germination may be influenced by respiratory factors, and alterations in ABA content and auxin distribution and responses. TOR (a serine/threonine-protein kinase) is likely at the centre of a regulatory hub controlling seed metabolism, maturation, and germination. Over-representation of the respiration-associated genes (ACO3 , PEPC3 , and D2HGDH ) was detected in non-dormant seed, suggesting differential energy supplies in the two seeds. Degradation of ABA biosynthesis and/or proper auxin signalling in the large seed may control germinability, and suppression of endoreduplication in the small seed may be a mechanism for cell differentiation and cell size determination.

Puerarin mitigates oxidative injuries, opening of mitochondrial permeability transition pores and pathological damage associated with liver and kidney in Xanthium strumarium-intoxicated rats.

In this study, the therapeutic effects of puerarin on Xanthium strumarium toxicity, which can develop in many species and does not have a specific antidote, were investigated. A single dose of 100 g/kg X. strumarium seeds was administered by gavage to female Sprague-Dawley rats, 6 h following which 200 mg/kg puerarin was administered by the same route, with puerarin administration being repeated daily at the same time. After completing the application, the blood, liver and kidney tissues of the rats were examined. Further, the biochemical parameters, glucose, MDA, GSH, SOD, mitochondrial Ca2+ and mitochondrial permeability transition pore (mPTP) opening levels, apoptotic factors (TUNEL, Bax and Bcl-2), ATP synthase and histopathological changes of the experimental rats were examined. The results revealed that while the administration of X. strumarium resulted in increased blood AST, ALT, ALP, LDH, CK, BUN and creatinine levels, it decreased glucose levels. In addition, it increased the MDA levels in the tissues and significantly increased the oxidative stress levels by decreasing the GSH levels and SOD activity. X. strumarium caused an increase in the mitochondrial Ca2+ and mPTP opening levels. Moreover, it increased the immunohistochemically determined ATP synthase expression and histopathologically identified necrotic liver cell death rates. Owing to its antioxidant properties and inhibitory effects on mPTP opening, puerarin administered for therapeutic purposes decreased the oxidative damage caused by X. strumarium toxicity, blood biochemical parameter levels, mitochondrial Ca2+ levels, mPTP opening, ATP synthase expression and the percentage of necrotic cells. Hence, the reduction in the liver and kidney damage in X. strumarium toxicity by puerarin indicates its potential use as an antidote for X. strumarium poisoning.

Metabolomic differences between invasive alien plants from native and invaded habitats.

Globalization facilitated the spread of invasive alien species (IAS), undermining the stability of the world's ecosystems. We investigated the metabolomic profiles of three IAS species: Chromolaena odorata (Asteraceae) Datura stramonium (Solanaceae), and Xanthium strumarium (Asteraceae), comparing metabolites of individual plants in their native habitats (USA), to their invasive counterparts growing in and around Kruger National Park (South Africa, ZA). Metabolomic samples were collected using RApid Metabolome Extraction and Storage (RAMES) technology, which immobilizes phytochemicals on glass fiber disks, reducing compound degradation, allowing long-term, storage and simplifying biochemical analysis. Metabolomic differences were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) of samples eluted from RAMES disks. Partial Least Squares-Discriminant Analysis (PLS-DA) of metabolomes of individual plants allowed statistical separation of species, native and invasive populations of each species, and some populations on the same continent. Invasive populations of all species were more phytochemically diverse than their native counterparts, and their metabolomic profiles were statistically distinguishable from their native relatives. These data may elucidate the mechanisms of successful invasion and rapid adaptive evolution of IAS. Moreover, RAMES technology combined with PLS-DA statistical analysis may allow taxonomic identification of species and, possibly, populations within each species.

Morphophysiological responses and tolerance mechanisms of Xanthium strumarium to manganese stress.

Effective phytoremediation of manganese (Mn) requires the careful selection of a species that has a relatively high manganese tolerance. Exploring the physiological mechanisms related to Mn stress responses is crucial for identifying and employing species for Mn phytoremediation. Xanthium strumarium is a species that can accumulate high levels of Mn, thus it is a candidate species for Mn-phytoremediation. To reveal the tolerance mechanisms of this species to manage Mn stress, the morphological, physiological, and biochemical responses of seedlings grown in water cultures under six different Mn concentrations were analyzed. The results showed that X. strumarium can accumulate high levels of Mn, even as plant growth was inhibited by rising Mn concentrations. Malondialdehyde (MDA) content increased and catalase (CAT) activity decreased along with the increased Mn concentrations, while soluble protein and proline content, as well as the superoxide dismutase (SOD) and peroxidase (POD) enzymes, all increased initially and then declined. The highest value of POD, SOD, soluble protein and proline all occurred at 5000 µM of Mn stress, which means that X. strumarium can adapt to low concentration of Mn stress. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) decreased, and the stomatal limitation (Ls) increased in response to Mn stress. Furthermore, water use efficiency (WUE) and intrinsic water use efficiency (WUEi) increased first under low concentration of Mn, and then reduced as the concentration of Mn increased. The maximum quantum efficiency of PSII photochemistry (Fv/Fm), efficiency of excitation capture by open PSII reaction centers (Fv'/Fm'), electron transport rate (ETR) declined as Mn concentration increased. In conclusion, the above results showed that X. strumarium can be effectively used for phytoremediation of Mn-contaminated soils.

Inconsistent intraspecific pattern in leaf life span along nitrogen-supply gradient.

PREMISE OF THE STUDY: Leaf life span (LLS) has long been hypothesized to plastically increase with decreasing nitrogen (N) supply from soil to maximize N retention, carbon assimilation, and fitness; however, accumulating evidence shows no consistent trend. The apparent inconsistencies are explained by a recent model that assumes LLS has a hump-shaped quadratic response to the N-supply gradient. The available evidence mostly originates from comparisons of LLS at only two levels of N availability, and the hypothesis remains unanswered. METHODS: We investigated LLS of two asteraceous forbs (Adenocaulon himalaicum and Xanthium canadense) experimentally grown at eight levels of N supply, which covered a range of N supply in their natural habitats. We additionally conducted a literature search to retrieve studies reporting LLS response along an N-supply gradient. KEY RESULTS: The LLS of neither species showed a hump-shaped response along the N-supply gradient. Past studies examining the LLS of an aquatic forb and terrestrial shrubs and trees along the N-supply gradient (more than four levels of N supply) also refuted the hypothesis. CONCLUSIONS: The LLS of a single species exhibited neither an increase nor a hump-shaped response to decreased N supply in a variety of life forms. Comparisons at only a few N levels are misleading with regard to LLS response to N supply.

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

Sesquiterpenes with TRAIL-resistance overcoming activity from Xanthium strumarium.

The ability of TRAIL to selectively induce apoptosis in cancer cells while sparing normal cells makes it an attractive target for the development of new cancer therapy. In search of bioactive natural products for overcoming TRAIL-resistance from natural resources, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionations of the extract of Xanthium strumarium led to the isolation of five sesquiterpene compounds (1-5). 11α,13-dihydroxanthinin (2) and 11α,13-dihydroxanthuminol (3) were first isolated from natural resources and xanthinosin (1), desacetylxanthanol (4), and lasidiol p-methoxybenzoate (5) were known compounds. All compounds (1-5) showed potent TRAIL-resistance overcoming activity at 8, 20, 20, 16, and 16 µM, respectively, in TRAIL-resistant AGS cells. Compounds 1 and 5 enhanced the levels of apoptosis inducing proteins DR4, DR5, p53, CHOP, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 and also decreased the levels of cell survival protein Bcl-2 in TRAIL-resistant AGS cells in a dose-dependent manner. Compound 1 also enhanced the levels of DR4 and DR5 proteins in a time-dependent manner. Thus, compounds 1 and 5 were found to induce both extrinsic and intrinsic apoptotic cell death. Compound 1 also exhibit TRAIL-resistance overcoming activity in DLD1, DU145, HeLa, and MCF7 cells but did not decrease viability in non-cancer HEK293 cells up to 8 µM.

Biomimetic synthesis: discovery of xanthanolide dimers.

Starting from xanthatin, the biomimetic synthesis of 4ß,5ß-epoxyxanthatin-1α,4α-endoperoxide, a novel monomeric xanthanolide, has been achieved. Moreover, four unprecedented xanthanolide dimers were synthesized by three different dimerizations of xanthatin, either in a head-to-head or head-to-tail fashion. Notably, these dimeric compounds were firstly identified as artifacts in the laboratory, and two of them, mogolides A and B, proved to be natural products present in the Xanthium mogolium Kitag plant.

In vitro cytotoxicity and in vivo acute and chronic toxicity of Xanthii Fructus and its processed product.

Xanthii Fructus (XF), the fruit of Xanthium sibiricum Patr., was used in the treatment of rhinitis and related nasal disease. Adverse effects of Xanthii Fructus are frequently reported these years. In the paper, in vitro renal cytotoxicity and in vivo acute and chronic toxicity researches of Xanthii Fructus (XF) and its processed product (processed Xanthii Fructus (PXF)) were carried out. Water extraction of XF displayed no cell membrane damage effects even in the highest concentration (100 µg/mL); however, it might affect the function of renal cell mitochondria. Acute toxicities were observed only in high and middle dosage groups. Fortunately, the single dose administration of XF or PXF was safe even at the highest daily dosage. Twelve-week chronic toxicity assays were performed in SD rats with low, middle, and high dosage. Notable changes in body weight and blood cell and BUN and Scr changes sporadically occurred in middle and high groups after the 9th week. Serum HA and HPCIII values were sustained increasing from the 4th week to the 8th week in Group V male rats, which indicated that the renal fibrosis risks still existed although no fibrosis was found in the pathological examination of the liver and kidney.

Testing a vapour-phase model of stomatal responses to humidity.

This study tests two predictions from a recently proposed model for stomatal responses to humidity and temperature. The model is based on water potential equilibrium between the guard cells and the air at the bottom of the stomatal pore and contains three independent variables: gs(0), Z and Θ. gs(0) is the value of stomatal conductance that would occur at saturating humidity and will vary among leaves and with CO2 and light. The value of Z is determined primarily by the resistance to heat transfer from the epidermis to the evaporating site and the value of Θ is determined primarily by the resistance to water vapour diffusion from the evaporating site to the guard cells. This leads to the two predictions that were tested. Firstly, the values of Z and Θ should be constant for leaves of a given species grown under given conditions, although gs(0) should vary among leaves and with light and CO2. And secondly, the ratio of Z to Θ should be higher in leaves having their stomata in crypts because the distance for heat transfer is greater than that for water vapour diffusion. Data from three species, Nerium oleander, Pastinaca sativum and Xanthium strumarium support these two predictions.

Quality assessment of Fructus xanthii based on fingerprinting using high-performance liquid chromatography.

Because almost every traditional Chinese medicine (TCM) is a multicomponent system, QC of TCMs always involves various difficulties. As a current popular quality assessment approach, focusing on qualitative and quantitative analysis of certain compounds contained in herbal medicine has been widely used for the sake of expediency rather than being a practical and realistic way. However, this method does not take the existence of other constituents into account. Comparatively, the chromatographic fingerprint of the components is a more suitable approach to holistically assess the quality of herbal drugs. Fructus xanthii is a well-known herbal drug listed in all editions of the Chinese Pharmacopoeia. However, there is no quality evaluation method given in its monograph, even for the above-mentioned expediency. This paper reports an HPLC fingerprinting method for quality evaluation of F. xanthii. The HPLC profiles of 27 batches of commercial samples were further analyzed using chemometric methods, including similarity evaluation and principal component analysis. As a result, the established HPLC fingerprint contained 23 characteristic peaks; therein, 13 peaks were unambiguously assigned by comparing their retention times and UV spectra with those of reference compounds, and five peaks were tentatively identified on the basis of their MS/MS fragmentation patterns and UV spectra. Moreover, it could be clearly observed that caffeoylquinic acid and its analogs predominate in F. xanthii. Except for three samples identified as outliers, 24 other commercial samples displayed similar HPLC profiles, indicating that the quality of the herbs from different markets is stable and consistent.

Growth and nitrogen use in Xanthium canadense grown in an open or in a dense stand.

Plants develop branches profusely when grown solitarily, while less so when grown in a dense stand. Such changes in architecture are associated with changes in dry mass allocation and nitrogen use. Here, we studied what traits in plant growth and nitrogen use were influenced by different light climates in the stand. Annual plants (Xanthium canadense) were grown solitarily or in a dense stand. Dry mass growth was analyzed as the product of the net assimilation rate (NAR) and leaf area (LA). Nitrogen use efficiency (NUE) was analyzed as the product of nitrogen productivity (NP) and the mean residence time (MRT) of nitrogen. These growth variables were further factorized into their components. Solitary plants maintained a high NAR, whereas plants in the dense stand decreased the NAR due to mutual shading. Plants in the dense stand developed a larger LA with a higher specific leaf area than solitary plants. Solitary plants had higher NUE due to higher NP. A temporal increase in NUE was attributed to the increase in MRT of nitrogen. Light climate was different between solitary and dense-stand plants, but they took up a comparable amount of nitrogen and used it differently in response to the given light climate. NUE was thus demonstrated to be a useful tool for analyzing the mechanism leading to different N use in plant growth.

Control of transpiration by radiation.

The terrestrial hydrological cycle is strongly influenced by transpiration--water loss through the stomatal pores of leaves. In this report we present studies showing that the energy content of radiation absorbed by the leaf influences stomatal control of transpiration. This observation is at odds with current concepts of how stomata sense and control transpiration, and we suggest an alternative model. Specifically, we argue that the steady-state water potential of the epidermis in the intact leaf is controlled by the difference between the radiation-controlled rate of water vapor production in the leaf interior and the rate of transpiration. Any difference between these two potentially large fluxes is made up by evaporation from (or condensation on) the epidermis, causing its water potential to pivot around this balance point. Previous work established that stomata in isolated epidermal strips respond by opening with increasing (and closing with decreasing) water potential. Thus, stomatal conductance and transpiration rate should increase when there is condensation on (and decrease when there is evaporation from) the epidermis, thus tending to maintain homeostasis of epidermal water potential. We use a model to show that such a mechanism would have control properties similar to those observed with leaves. This hypothesis provides a plausible explanation for the regulation of leaf and canopy transpiration by the radiation load and provides a unique framework for studies of the regulation of stomatal conductance by CO(2) and other factors.

In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid "cycle" in illuminated leaves.

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, (13)C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA "cycle" does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.

Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus Xanthii from different populations in China.

Fructus Xanthii (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. For the effective quality control of its medicinal values, reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on UV spectrophotometry, HPLC fingerprinting and inter-simple sequence repeat (ISSR) molecular markers were carried out, involving 16 Xanthium populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the 16 Xanthium populations, and they were classified to three chemotypes by hierarchical clustering analysis. Abundant genetic diversity was detected among the Xanthium populations, which were also clustered into three groups based on their ISSR data and varied according to different species. Combining the genetic divergence and chemical differences showed an important result that, in the two chemotypes, the higher contents of total phenolic acids (TPA) in Fructus Xanthii showed greater genetic diversity (I). We suggest that genetic diversity affects the contents of TPA. Since variable phenolic acid contents may affect therapeutic efficacy, it is important to point out that combining the use of genetic base with chemotype will help control the favourable chemotypes and breed new cultivars with more desirable chemical constituents.

Leaf hydraulic conductivity and stomatal responses to humidity in amphistomatous leaves.

The response of stomata to changes in humidity for a single surface of an amphistomatous leaf was investigated in Xanthium strumarium and Vicia faba using gas exchange and direct observation of stomatal apertures. The stomatal response to humidity for a given surface was found to be the same whether or not the humidity for the opposite surface was changed concurrently. Stomata on the surface for which humidity was constant showed no response to changes in humidity for the opposite surface. Despite large changes in epidermal turgor on the surface for which humidity was changed, there was no change in epidermal turgor for the surface with constant humidity. Measurements of transpiration and epidermal turgor as functions of the mole fraction gradient of water between leaf and air were used to calculate a value for leaf hydraulic resistance. The results suggest that in these species, the mechanism for the stomatal response to humidity resides in the epidermis or the mesophyll very close to the epidermis, and that most of the hydraulic resistance of the leaf occurs between the xylem and the evaporating sites.

Leaf lifespan and lifetime carbon balance of individual leaves in a stand of an annual herb, Xanthium canadense.

Leaf lifespan in response to resource availability has been documented in many studies, but it still remains uncertain what determines the timing of leaf shedding. Here, we evaluate the lifetime carbon (C) balance of a leaf in a canopy as influenced by nitrogen (N) availability. Stands of Xanthium canadense were established with high-nitrogen (HN) and low-nitrogen (LN) treatments and temporal changes of C gain of individual leaves were investigated with a canopy photosynthesis model. Daily C gain of a leaf was maximal early in its development and subsequently declined. Daily C gain at shedding was nearly zero in HN, while it was still positive in LN. Sensitivity analyses showed that the decline in the daily C gain resulted primarily from the reduction in light level in HN and by the reduction in leaf N in LN. Smaller leaf size in LN than in HN led to higher light levels in the canopy, which helped leaves of the LN stand maintain for a longer period. These results suggest that the mechanism by which leaf lifespan is determined changes depending on the availability of the resource that is most limiting to plant growth.

Effect of monosodium methanarsonate application on cuticle wax content of cocklebur and cotton plants.

Leaf cuticle waxes were extracted from monosodium methanearsonate (MSMA)-resistant (R) and -susceptible (S) common cocklebur (Xanthium strumarium L.) and cotton (Gossypium hirsutum L.) plants at 0, 3, 5, and 7 days after treatment (DAT) following 1x and 2x MSMA applications. Wax constituents were analyzed by gas chromatography (GC) with flame ionization detection and compared to alkane and alcohol standards of carbon lengths varying from C21 to C30. Differences in waxes were calculated and reported as change per ng mm2-1. Tricosane (C23) was found to increase following MSMA applications. All other alkanes decreased by 7 DAT, with some showing a linear effect over time in the R-cocklebur. Alcohol constituents were also observed to decrease by 7 DAT. Total arsenic in the extracted wax fraction was determined, with greatest quantities detected in the R-cocklebur. Wax changes are not believed to play a role in cotton tolerance, since changes in cuticle concentrations were minimal. Cocklebur resistance to MSMA is not due to cuticle constituents; the wax changes are a secondary effect in response to herbicide application.