Analysis of cellulose synthase activity in Arabidopsis using spinning disk microscopy.

We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings.

Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content.

Brassinosteroids Influence Arabidopsis Hypocotyl Graviresponses through Changes in Mannans and Cellulose.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base.

Betacyanins and Betaxanthins in Cultivated Varieties of Beta vulgaris L. Compared to Weed Beets.

There are 11 different varieties of Beta vulgaris L. that are used in the food industry, including sugar beets, beetroots, Swiss chard, and fodder beets. The typical red coloration of their tissues is caused by the indole-derived glycosides known as betalains that were analyzed in hypocotyl extracts by UV/Vis spectrophotometry to determine the content of betacyanins (betanin) and of betaxanthins (vulgaxanthin I) as constituents of the total betalain content. Fields of beet crops use to be also infested by wild beets, hybrids related to B. vulgaris subsp. maritima or B. macrocarpa Guss., which significantly decrease the quality and quantity of sugar beet yield; additionally, these plants produce betalains at an early stage. All tested B. vulgaris varieties could be distinguished from weed beets according to betacyanins, betaxanthins or total betalain content. The highest values of betacyanins were found in beetroots 'Monorubra' (9.69 mg/100 mL) and 'Libero' (8.42 mg/100 mL). Other beet varieties contained less betacyanins: Sugar beet 'Labonita' 0.11 mg/100 mL; Swiss chard 'Lucullus,' 0.09 mg/100 mL; fodder beet 'Monro' 0.15 mg/100 mL. In contrast with weed beets and beetroots, these varieties have a ratio of betacyanins to betaxanthins under 1.0, but the betaxanthin content was higher in beetcrops than in wild beet and can be used as an alternative to non-red varieties. Stability tests of selected varieties showed that storage at 22 °C for 6 h, or at 7 °C for 24 h, did not significantly reduce the betalain content in the samples.

Chemical Analysis of Lepidium meyenii (Maca) and Its Effects on Redox Status and on Reproductive Biology in Stallions †.

The present study was conducted to assess the chemical composition of Yellow Maca (Lepidium meyenii) and its biological activity on stallions following oral administration of hypocotyl powder. Maca was subjected to methanolic extraction and the chemical analysis was carried out by LC-MS-QTOF (liquid chromatography-mass spectrometry). Our results showed that Maca contains some effective antioxidants, a high percentage of glucosinolates, and other important components with a high antioxidant capacity. To evaluate the plant biological activity in stallions fed with Maca powder for 60 days, the redox status and some reproductive parameters were investigated. Blood and semen samples were collected at 0, 30, 60, and 90 days from the beginning of this study. Blood samples showed a decrease of the reactive oxygen metabolites, evaluated by d-ROMs test, and an increase of the antioxidant barrier in terms of biological antioxidant potential (BAP test), powerful oxidant capacity (OXY-Adsorbent test), and thiols evaluation (-SHp test). Furthermore, semen samples showed a positive trend during Maca administration in the following parameters: ejaculate volumes and sperm concentrations, total and progressive motility, and acrosome integrity.

Zinc and Cadmium Mapping in the Apical Shoot and Hypocotyl Tissues of Radish by High-Resolution Secondary Ion Mass Spectrometry (NanoSIMS) after Short-Term Exposure to Metal Contamination.

Zinc (as an essential phytonutrient) and cadmium (as a toxic but readily bioavailable nonessential metal for plants) share similar routes for crossing plant biomembranes, although with a substantially different potential for translocation into above-ground tissues. The in situ distribution of these metals in plant cells and tissues (particularly intensively-dividing and fast-growing areas) is poorly understood. In this study, 17-day-old radish (Raphanus sativus L.) plants grown in nutrient solution were subjected to short-term (24 h) equimolar contamination (2.2 µM of each 70Zn and Cd) to investigate their accumulation and distribution in the shoot apex (leaf primordia) and edible fleshy hypocotyl tissues. After 24-h exposure, radish hypocotyl had similar concentration (in µg/g dry weight) of 70Zn (12.1 ± 1.1) and total Cd (12.9 ± 0.8), with relatively limited translocation of both metals to shoots (concentrations lower by 2.5-fold for 70Zn and 4.8-fold for Cd) as determined by inductively-coupled plasma mass spectrometry (ICP-MS). The in situ Zn/Cd distribution maps created by high-resolution secondary ion mass spectrometry (NanoSIMS, Cameca, Gennevilliers, France) imaging corresponded well with the ICP-MS data, confirming a similar pattern and uniform distribution of 70Zn and Cd across the examined areas. Both applied techniques can be powerful tools for quantification (ICP-MS) and localisation and visualisation (NanoSIMS) of some ultra-trace isotopes in the intensively-dividing cells and fast-growing tissues of non-metalophytes even after short-term metal exposure. The results emphasise the importance of the quality of (agro)ecosystem resources (growing media, metal-contaminated soils/waters) in the public health risk, given that, even under low contamination and short-term exposure, some of the most toxic metallic ions (e.g., Cd) can relatively rapidly enter the human food chain.

Production of a new mucilage compound in Lepidium sativum callus by optimizing in vitro growth conditions.

The mucilage in Lepidium sativum L. is considered a biologically active compound with diverse medicinal properties. Different explants (hypocotyls and leaf) were transferred to Murashige and Skoog (MS) medium supplemented with twelve different plant growth regulator combinations under two different incubations (light and dark). The best mucilage production from callus (36.76% g g-1 dry weight) was obtained in the MS medium supplemented with 1 mg L-1 of 2, 4-D and 2 mg L-1 of BAP under the light condition. The mucilage produced by callus culture was nearly three times more than the mucilage yield of the seeds. The glucose, arabinose + mannose and galactose were 43.4 (mg g-1 DW), 195.3 (mg g-1 DW) and 86.2 (mg g-1 DW) in the mucilage originated from seed, callus leaf and callus hypocotyl, respectively. The present study proposes an efficient method for producing large scales of mucilage with a favorable sugar aimed at food or pharmaceutical industries.

Dynamic changes in polyphenol compounds, antioxidant activity, and PAL gene expression in different tissues of buckwheat during germination.

BACKGROUND: There is a growing interest in buckwheat germination regarding the improvement of its health benefits. The aims of this study were to evaluate the effects of germination on polyphenol compounds, antioxidant activity, and phenylalanine ammonia-lyase (PAL) gene expression in different tissues (cotyledon, hypocotyl, and radicle) of buckwheat sprouts during germination for 12 days, as well as to investigate their interactions. RESULTS: Total polyphenol and total flavonoid contents, antioxidant activity, main polyphenol components, and PAL gene expression significantly increased during germination. On day 12, the rutin content in cotyledons was elevated to 88.6 g kg-1 , which was 7.7-times and 39.4-times compared to those in buckwheat seeds and radicles, respectively. Meanwhile, chlorogenic acid in hypocotyls reached 7.84 g kg-1 , which was 36.3-fold higher than those in radicles. However, the PAL gene showed the highest expression in radicles. CONCLUSION: Present results showed that polyphenol compounds mainly accumulated in cotyledons and hypocotyls. There was a negative correlation between polyphenol compounds and PAL gene expression. The discrepancy suggested that polyphenol compounds might experience transportation within buckwheat sprouts. The study could provide useful information for further application of buckwheat in functional foods, and revelation of the correlation between bioactive components and related gene expressions. © 2018 Society of Chemical Industry.

Combinational effects of non n-Hexane Fractions of ant-plant (Myrmecodia tuberosa Jack) hypocotyl with doxorubicin against lymphocyte and cancer cells.

Doxorubicin is widely used as a chemotherapeutic drug despite having many side effects. It may cause the dysfunction of macrophage, decreasing proliferation of lymphocytes, decreasing CD4+/CD8+ ratio and inducing hepatotoxicity. Doxorubicin inhibits the growth of Vero, HeLa, and T47D cell lines, and also induces a resistance of MCF-7 cells. Previous studies showed that ethanolic extract and ethyl acetate fraction of ant-plant (Myrmecodia tuberose Jack) hipocotyl could increase macrophage phagocytosis activity and lymphocyte proliferation in vitro. Therefore, antplant is a potential immune stimulator. Combinational treatment of non n-hexane fraction (NHF) of ant-plant with doxorubicin did not affect the doxorubicin's potency. Nevertheless, increased lymphocyte viability induced by doxorubicin in varied dosages of NHF that lethal to HeLa, MCF-7 and T47D cells. Moreover, on Vero cells, doxorubicin became less toxic when induced together with NHF. Thus, NHF of ant-plant is potential to be proposed as doxorubicin co-chemotherapeutic agent against cancer cells.

Structure based in silico identification of potentially non-steroidal brassinosteroids mimics.

Brassinosteroids (BRs) are a class of plant steroid hormones that play indispensable roles in cell elongation, division and plant development. To date, the numerous synthesis of BRs analogs and structure-activity relationship investigations have clearly revealed the key substituent groups relevant to the steroidal activity of BRs. However, due to the limited chemical space studied, the efforts for alternative non-steroidal compounds have produced no remarkable results. To identify potentially non-steroidal BR mimics in this study, vital interacting pharmacophore features were extracted starting from several complex structures of BRs that bound with the receptor Brassinosteroid-Insentive 1 (BRI1) and co-receptor BRI1-associated kinase 1 (BAK1), which were characterized and merged into one comprehensive pharmacophore model. In silico screening of a commercial compound database was carried out by combing pharmacophore modeling, molecular docking and visual analysis. Finally, six non-steroidal molecules were identified and subjected to the in vivo radish hypocotyl elongation assay. As a positive control, the hypocotyls elongation for the naturally most active BR brassinolide (BL) is 152 ± 3% at 100 nM. Moreover, two candidates (4 and 6) show good BRs-like activity with the hypocotyls elongation of 143 ± 1% and 128 ± 3% at the same dose, respectively. Most remarkably, compounds 4 and 6, which have different structures, are predicted to share similar binding modes and proven to exhibit potential BRs-like activity. The two compounds obtained could be valuable leads for the development of BRs-like plant growth regulators.

Organ-Specific Proteomic Analysis of Early Vegetative Stage Soybean.

BACKGROUND: Soybean is a nutritionally important source of proteins and oils. Organspecific analyses of the plants provided valuable information about physiological changes occurring in particular organ. However, studies on the vegetative stage soybean during the growth and development are lacking. Analyzing the growth stages of soybean at proteomic level is particularly important for understanding the growth dynamics. OBJECTIVES: The main objective of this study was to investigate the organ-specific growth pattern of proteins during early growth stage. METHODS: Soybean (Glycine max L. cv Enrei) seeds were sown in each seedling case for 10 days and roots, hypocotyls, and leaves were collected. Proteins were extracted and analyzed by nano-liquid chromatography mass spectrometry. Mole percent abundance was calculated using emPAI values. To determine the functional role of the proteins identified in the MS analysis, functional categorization was performed using MapMan bin codes. Visualization of protein abundance ratio was performed using MapMan software. Enzyme activity and quantitative reverse transcription-polymerase chain reaction analyses were performed. RESULTS: A total of 357, 360, and 392 proteins were identified in root, hypocotyl, and leaf of vegetative stage soybean, respectively. Proteins related to stress, cell organization, transport, signaling, and mitochondrial electron transport chain decreased in root, hypocotyl, and leaf. Proteins related to protein metabolism, glycolysis, and cell wall were comparable in root and hypocotyl; however, in leaf, glycolysis and cell wall related proteins were decreased. Aldehyde dehydrogenase was decreased in abundance and activity in hypocotyl and leaf as compared to root. Major latex proteins 43 and 423 changed in abundance in an organ-specific manner. The mRNA expression level of major latex proteins exhibited a differential expression in the hypocotyl of soybean during flooding stress and recovery. CONCLUSION: Our results suggest that aldehyde dehydrogenase and major latex proteins play key roles in growth of soybean in an organ-specific way.

Isolation of the Cell Wall.

This chapter describes a method allowing the purification of the cell wall for studying both polysaccharides and proteins. The plant primary cell wall is mainly composed of polysaccharides (90-95 % in mass) and of proteins (5-10 %). At the end of growth, specialized cells may synthesize a lignified secondary wall composed of polysaccharides (about 65 %) and lignin (about 35 %). Due to its composition, the cell wall is the cellular compartment having the highest density and this property is used for its purification. It plays critical roles during plant development and in response to environmental constraints. It is largely used in the food and textile industries as well as for the production of bioenergy. All these characteristics and uses explain why its study as a true cell compartment is of high interest. The proposed method of purification can be used for large amount of material but can also be downscaled to 500 mg of fresh material. Tools for checking the quality of the cell wall preparation, such as protein analysis and microscopy observation, are also provided.

Proteomic analysis of soybean root including hypocotyl during recovery from drought stress.

UNLABELLED: Soybean is a nutritionally important crop that exhibits reductions in growth and yield under drought stress. To investigate soybean responses during post-drought recovery, a gel-free proteomic technique was used. Two-day-old soybeans were treated with drought stress for 4days and recovered for 4days. Root including hypocotyl was collected during the drought treatment and recovery stage. Seedling growth was suppressed by drought stress, but recovered following stress removal. The malondialdehyde content increased under drought stress, but decreased during the recovery stage. A total of 792 and 888 proteins were identified from the control and recovering seedlings, respectively. The identified proteins were related to functional categories of stress, hormone metabolism, cell wall, secondary metabolism, and fermentation. Cluster analysis indicated that abundances of peroxidase and aldehyde dehydrogenase were highly changed in the seedlings during the post-drought recovery. The activity of peroxidase decreased under drought conditions, but increased during recovery. In contrast, the activity of aldehyde dehydrogenase was increased in response to drought stress, but decreased during the recovery stage. These results suggest that peroxidase and aldehyde dehydrogenase play key roles in post-drought recovery in soybean by scavenging toxic reactive oxygen species and reducing the load of harmful aldehydes. BIOLOGICAL SIGNIFICANCE: Post-drought recovery response mechanisms in soybean root including hypocotyl were analyzed using gel-free proteomic technique. A total of 643 common proteins between control and drought-stressed soybeans changed significantly in abundance over time. The proteins that changed during post-drought recovery were assigned to protein, stress, hormone metabolism, secondary metabolism, cell wall, redox, and glycolysis categories. The analysis revealed that peroxidase and aldehyde dehydrogenase were increased in protein abundance under drought stress. The enzyme activity of peroxidase decreased under drought but increased during recovery. The activity of aldehyde dehydrogenase was increased under drought stress but decreased during recovery stage. Peroxidase and aldehyde dehydrogenase reduce the toxic reactive oxygen species and aldehydes from the plant, respectively, and help to recover from drought stress. The study provides information about post-drought recovery mechanism in soybean.

Light piping driven photosynthesis in the soil: Low-light adapted active photosynthetic apparatus in the under-soil hypocotyl segments of bean (Phaseolus vulgaris).

Photosynthetic activity was identified in the under-soil hypocotyl part of 14-day-old soil-grown bean plants (Phaseolus vulgaris L. cv. Magnum) cultivated in pots under natural light-dark cycles. Electron microscopic, proteomic and fluorescence kinetic and imaging methods were used to study the photosynthetic apparatus and its activity. Under-soil shoots at 0-2cm soil depth featured chloroplasts with low grana and starch grains and with pigment-protein compositions similar to those of the above-soil green shoot parts. However, the relative amounts of photosystem II (PSII) supercomplexes were higher; in addition a PIP-type aquaporin protein was identified in the under-soil thylakoids. Chlorophyll-a fluorescence induction measurements showed that the above- and under-soil hypocotyl segments had similar photochemical yields at low (10-55µmolphotonsm(-2)s(-1)) light intensities. However, at higher photon flux densities the electron transport rate decreased in the under-soil shoot parts due to inactivation of the PSII reaction centers. These properties show the development of a low-light adapted photosynthetic apparatus driven by light piping of the above-soil shoot. The results of this paper demonstrate that the classic model assigning source and sink functions to above- and under-soil tissues is to be refined, and a low-light adapted photosynthetic apparatus in under-soil bean hypocotyls is capable of contributing to its own carbon supply.

Characteristic fingerprinting based on macamides for discrimination of maca (Lepidium meyenii) by LC/MS/MS and multivariate statistical analysis.

BACKGROUND: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS). RESULTS: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification. CONCLUSION: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry.

Four New Cyclohexylideneacetonitrile Derivatives from the Hypocotyl of Mangrove (Bruguiera gymnorrhiza).

Four new cyclohexylideneacetonitrile derivatives 1-4, named menisdaurins B-E, as well as three known cyclohexylideneacetonitrile derivatives--menisdaurin (5), coclauril (6), and menisdaurilide (7)--were isolated from the hypocotyl of a mangrove (Bruguiera gymnorrhiza). The structures of the isolates were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-7 showed anti-Hepatitis B virus (HBV) activities, with EC50 values ranging from 5.1 ± 0.2 µg/mL to 87.7 ± 5.8 µg/mL.

[Study on Chemical Constituents from Hypocotyls of Mangrove Bruguiera gymnorrhiza].

OBJECTIVE: To study the chemical constituents from the hypocotyls of mangrove Bruguiera gymnorrhiza. METHODS: The chemical constituents were isolated and purified by recrystallization, silica gel column chromatography and semi-preparative HPLC. Their structures were identified by spectroscopic analysis and comparison with literatures. RESULTS: Seven compounds were isolated and their structures were identified as 3-ß-(Z)-coumaroyllupeol (1), dioslupecin (2), cholesterol (3), menisdaurillide (4), aquilegiolide (5) vomifoliol (6) and roseoside II (7). CONCLUSION: Compounds 1,2 and 4 - 7 are isolated from this plant for the first time.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Bioactive maca (Lepidium meyenii) alkamides are a result of traditional Andean postharvest drying practices.

Maca, Lepidium meyenii Walpers (Brassicaceae), is an annual herbaceous plant native to the high plateaus of the Peruvian central Andes. Its underground storage hypocotyls have been a traditional medicinal agent and dietary staple since pre-Columbian times. Reported properties include energizing and fertility-enhancing effects. Published reports have focused on the benzylalkamides (macamides) present in dry hypocotyls as one of the main bioactive components. Macamides are secondary amides formed by benzylamine and a fatty acid moiety, with varying hydrocarbon chain lengths and degree of unsaturation. Although it has been assumed that they are usually present in fresh undamaged tissues, analyses show them to be essentially absent from them. However, hypocotyls dried by traditional Andean postharvest practices or industrial oven drying contain up to 800µgg(-1) dry wt (2.3µmolg(-1) dry wt) of macamides. In this study, the generation of macamides and their putative precursors were studied during nine-week traditional drying trials at 4200m altitude and in ovens under laboratory conditions. Freeze-thaw cycles in the open field during drying result in tissue maceration and release of free fatty acids from storage and membrane lipids up to levels of 1200µgg(-1) dry wt (4.3µmolg(-1) dry wt). Endogenous metabolism of the isothiocyanates generated from glucosinolate hydrolysis during drying results in maximal benzylamine values of 4300µgg(-1) dry wt (40.2µmolg(-1) dry wt). Pearson correlation coefficients of the accumulation profiles of benzylamine and free fatty acid to that of macamides showed good values of 0.898 and 0.934, respectively, suggesting that both provide sufficient substrate for amide synthesis during the drying process.