Alkamides: a new class of plant growth regulators linked to humic acid bioactivity
The use of humic substances as plant biostimulants has been increasingly attracting farmers and stunning researchers. The ability of these substances to enhance root growth by changing root architecture is often linked to their hormonal activities, such as auxin effects and nitric oxide production. Humeomics accesses the molecular constituents of humic substances, revealing the importance of alkyl components because of their conformations and chemical activities. Here, we describe the alkamides present in humic acids and compare their bioactivities using plasma membrane H+-ATPase activity level as a biochemical marker.
Humic acids isolated from vermicompost were analyzed using 13C and 15N nuclear magnetic resonance spectroscopy. The unbound fraction was extracted with ethyl acetate and submitted to gas chromatography coupled to mass spectrometry to detect the presence of N-isopropyldecanamide. We synthesized N-isopropyldecanamide and treated maize seedlings for 7 and 15 days with different concentrations. The root growth and plasma membrane H+-ATPase activity were monitored. Nitric oxide accumulation in the lateral roots was imaged using 4,5-diaminofluorescein diacetate. The results were compared with those obtained for seedlings treated with humic acids isolated from vermicompost.
The amide functional group produced the only nitrogen signal in the 15N humic acid resonance spectrum and similar alkamide moieties were found in the unbound humic extract through comparisons using gas chromatography coupled to mass spectrometry. The synthesis of N-isopropyldecanamide had few steps and produced a high yield (86%). The effects of N-isopropyldecanamide on root growth were concentration dependent. High concentrations (10−4 M) enhanced root growth after 15 day of diminishing shoot biomass. However, low concentrations (10−8 M and 10−6 M) promoted root growth at 7 and 15 days, similar to the humic acid-induced plasma membrane H+-ATPase activity. Both N-isopropyldecanamide and humic acids enhanced nitric oxide accumulation during lateral root emergence.
KeywordsAffinins Small lipids Hormone-like effects Plant growth regulators Humic substances
gas chromatography coupled to mass spectrometry with ionization by electron impact
- PM H+-ATPase
plasma membrane proton ATPase
humic acids isolated from cattle manure vermicompost
- DAF-2 DA
Humic substances (HS), constituting a category of plant biostimulants, can be used directly on plants in low concentration to enhance nutrient uptake, plant growth and yield . The effects of HS on plant physiology and metabolism have been attributed to their putative hormone activities , which are mainly auxin-like [3, 4, 5, 6] because other plant hormones, such as gibberellins, cytokinins, nitric oxide (NO) and ethylene, are found in insignificant concentrations in soils and HSs [7, 8, 9, 10, 11, 12, 13, 14]. Curiously, the main nitrogen species often related to humic structures by 15N nuclear magnetic resonance (NMR) spectroscopy are present in amide functional groups, as revealed by the dominant peaks between − 245 and − 260 ppm [15, 16]. Furthermore, the recalcitrant or hydrophobic nature of HS was previously related to their bioactivities [17, 18, 19, 20].
In the early 2000s, a new class of plant growth regulators, called affinins, was described by Ramirez-Chávez et al. . Alkamides are secondary metabolites comprising over 200 related compounds having a general structure that originates from the condensation of an unsaturated fatty acid and an amine. Alkamides promote lateral root formation and root hair elongation, which are similar to the effects produced by auxins, but the ability of the root system to respond to affinins is independent of auxin signaling . The mechanisms through which the alkamides affect particular signal transduction cascades that modify root growth and differentiation are unknown, but the involvement of cytokinin receptors and NO production have been reported during root development [22, 23]. In addition, Morquecho-Contreras et al.  related the structures of alkamides, such as N-isopropyldecanamide, to the bacterial quorum-sensing signals, N-acyl-l-homoserine lactones. These compounds participate in cell-to-cell signaling, usually referred to as quorum sensing, which is a fundamental step in endophytic bacteria biofilm formation and host colonization . Considering the effects of biostimulants manufactured using humic acids (HA) and plant growth-promoting bacteria on plant physiology [25, 26, 27], as well as the wide distribution of these lipids in unbound soil fractions and compost humeomes [28, 29, 30, 31, 32], we determined whether (i) this class of compounds was linked to HA bioactivity levels and whether (ii) alkamides are present in the supramolecular structure of HA.
HA change the cellular electrical environment by enhancing H+ efflux . Proton pump activity levels can be used as biochemical markers of HA bioactivity . We synthesized N-isopropyldecanamide, an abundant plant alkamide, and used different concentrations to treat maize seedlings. The root growth and the number of root mitotic sites were measured, as well as the effects on the plasma membrane H+-ATPase activity level and NO production. The unbound HA fraction from cattle manure vermicompost was obtained using an organic solvent, and N-isopropyldecanamide was isolated from the humic supramolecular assembly. The alkamide structures in the HA were identified using a retention time comparison and mass fragmentation analysis.
Materials and methods
Synthesis of N-isopropyldecanamide
A solution of 29.0-mmol decanoic acid (5.0 g) in 54.8-mmol thionyl chloride (4.0 mL) was stirred and heated to reflux for 12 h. The excess thionyl chloride was removed by distillation, and the residue containing the decanoyl chloride was used in the next step without purification. A solution of 58.7-mmol isopropylamine (5 mL) in hexane (10 mL) was added dropwise to a stirred solution of decanoyl chloride in hexane (20 mL). During this period, the temperature of the reaction was maintained at 0–5 °C. Afterward, it was stirred at room temperature for 5 h. The salts were removed by filtration and washed twice with 20 mL H2O. The organic layer was dried and evaporated in a vacuum, and the resulting solid was purified by recrystallization from ethyl ether to yield N-isopropyldecanamide as a white solid (5.3 g, 86% yield) with m.p at 47 °C. The characterization of N-isopropyldecanamide was performed using NMR and gas chromatography coupled with mass spectroscopy (GC–MS) experiments. The 1H and 13C NMR spectra were recorded on a Jeol 400 instrument (1H: 400 MHz and 13C: 100 MHz; Tokyo, Japan) with TMS as the internal standard. Electron ionization (EI) mass spectra were obtained using a GC–MS Shimadzu QP5050A instrument at 70 eV. A DB-5 capillary column (30 m, 0.25 nm i.d.) was used with a heating rate of 15 °C min−1 from 50 to 230 °C. The injector temperature was set at 200 °C.
HA extraction and its chemical characterization
The HA used in this study were isolated from the vermicompost of cattle manure. HA were obtained according to the classical method of extraction, isolation and purification described on the Web page of the International Humic Substances Society (www.ihss.gated.edu). After freeze drying by lyophilization, the carbon content was analyzed using dry combustion (CHN analyzer Perkin Elmer series 2400, Norwalk, CT, USA). The chemical nature of the HA was accessed by cross-polarization magic angle spinning (CP/MAS) 13C and 15N NMR. The spectrum was acquired from the solid sample using a Bruker Avance 300 MHz (Bruker, Karlsruhe, Germany) equipped with a 4-mm wide bore MAS probe operating at a 13C-resonating frequency of 75.47 MHz. The 13C spectrum was integrated over the chemical shift (ppm) resonance intervals of 0–46 ppm (alkyl C, mainly CH2 and CH3 sp3 carbons), 46–65 ppm (methoxy and N alkyl C from OCH3, C–N and complex aliphatic carbons), 65–90 ppm (O-alkyl C, such as alcohols and ethers), 90–108 ppm (anomeric carbons in carbohydrate-like structures), 108–145 ppm (phenolic carbons), 145–160 ppm (aromatic and olefinic sp2 carbons), 160–185 ppm (carboxyl, amides and esters) and 185–225 ppm (carbonyls). The unbound fraction associated with HA was extracted from 100 mg of sample suspended in 1 mL of ethyl acetate at a pH previously adjusted to 11.0 with 1-M NaOH by stirring for 24 h at room temperature. The supernatant was separated by centrifugation (15 min, 3500×g), and the aliquot was injected into a Shimadzu QP5050A GC–MS (Tokyo, Japan) at 70 eV using a DB-5 capillary column (30 m; 0.25 nm d.i.) at 15 °C min−1 from 50 to 230 °C. The sample was injected at 200 °C.
Plant growth and HA treatment
Maize seeds (Zea mays L., var UENF 506) were surface sterilized by soaking in 0.5% NaClO for 30 min, rinsed and then soaked in water for 6 h. Afterward, the seeds were sown on wet filter paper and germinated in the dark at 28 °C. In the first experiment, 4-day-old maize seedlings with ~ 1 cm roots were transferred into a solution containing 2 mM CaCl2 with or without 20 mg CAH L−1 extracted from earthworm compost or 10−4, 10−6 or 10−8 M N-isopropyldecanamide. A minimal medium (2 mM CaCl2) was used to avoid any interference by nutrient constituents that could act synergistically with HA on plant growth and metabolism. In the second experiment, 4-day-old maize seedlings were transferred to Leonard pots containing sterile sand. On the first day, 500 mL half-strength Hoagland’s solution plus 20 mg C of HA or 10−4, 10−6 or 10−8 M N-isopropyldecanamide was added. The nutrient solution without HA or N-isopropyldecanamide was changed weekly. The roots were collected from 7- to 15-day-old seedlings in the first and second assays, respectively.
Root growth measurements
Root lengths and areas were measured using a Delta-T Scan software image analyzer (Delta-T Devices, Ltd, Cambridge, England). Other samples of root seedlings were collected and used in additional experiments.
Frequency of sites of lateral root emergence
The entire root systems were washed in water and cleaned by boiling at 75 °C for 20 min in 0.5% KOH. Afterward, root samples were rinsed in water and stained with a hematoxylin solution for 14 h in the dark. They were then rinsed in water and destained in 80% lactic acid at 75 °C for 30 to 90 s. Individual entire roots were transferred to Petri plates containing water and observed with a stereoscopic microscope to evaluate the number of visible mitotic sites on the root tissue. The hematoxylin stock solution contained 1 g hematoxylin, 0.5 g ferric ammonium sulfate and 50 mL 45% acetic acid, and it was stored in the dark at room temperature. Stains were prepared by diluting the stock solution 40-fold in water.
Plasma membrane (PM)-enriched vesicles
The PM-enriched vesicles were isolated from roots using differential centrifugation. Briefly, ~ 15 g (fresh weight) of maize roots was homogenized using a mortar and pestle in 30 mL of ice-cold buffer containing 250-mM sucrose, 10% (w/v) glycerol, 0.5% (w/v) PVP (40 kDa), 2-mM EDTA, 0.5% (w/v) BSA and 0.1-M Tris–HCl buffer at pH 8.0. Just prior to use, 150-mM KCl, 2-mM DTT and 1-mM PMSF were added to the buffer. The homogenate was strained through four layers of cheesecloth and centrifuged at 8000×g for 10 min. The supernatant was centrifuged once again at 8000×g for 10 min and then at 100,000×g for 40 min. The pellet was resuspended in a small volume of ice-cold buffer containing 10-mM Tris–HCl (pH 7.6), 10% (v/v) glycerol, 1-mM DTT and 1-mM EDTA. The suspension containing PM vesicles was layered over a 20%/30%/42% (w/w/w) discontinuous sucrose gradient that contained, in addition to sucrose, 10-mM Tris–HCl (pH 7.6), 1-mM DTT and 1-mM EDTA. After centrifugation at 100,000×g for 3 h in a swinging bucket, the vesicles at the interface between 30 and 42% sucrose were collected, diluted with three volumes of ice-cold water and centrifuged at 100,000×g for 40 min. The pellet was resuspended in a buffer containing 10-mM Tris–HCl (pH 7.6), 10% (v/v) glycerol, 1 mM DTT and 1 mM EDTA. The vesicles were either used immediately or frozen in liquid N2 and stored at − 70 °C until use. Protein concentrations were determined using Lowry’s method .
Plasma membrane H+-ATPase hydrolysis
The hydrolytic H+-ATPase activity levels in the PM-enriched vesicles were determined colorimetrically by measuring the release of Pi . Between 70 and 90% of the PM vesicle’s’ ATPase activity, measured at pH 6.5, was inhibited by vanadate (0.1 mM), a very effective inhibitor of the PM P-type H+-ATPase. The assay medium consisted of 1-mM ATP–BTP, 5-mM MgSO4, 10-mM MOPS–BTP (pH 6.5), 100-mM KCl, 0.2-mM Na2MoO4 and 0.05 mg mL−1 vesicle protein. In the experiments, ATPase activity was measured at 30 °C, with and without vanadate, and the difference between the two measurements was attributed to the PM H+-ATPase.
H+-pumping by PM H+-ATPase
The electrochemical H+-gradient generated by the H+-ATPase was estimated from the initial quenching rate of the fluorescent pH probe 9-amino-6-chloro-2-methoxyacridine (415/485 nm excitation/emission) and expressed in percentage quenching per min. The assay medium contained 10-mM HEPES–KOH (pH 6.5), 100-mM KCl, 3-mM MgCl2, 2.5-μM 9-amino-6-chloro-2-methoxyacridine and 0.05 mg L−1 PM vesicles protein. The reaction was triggered by the addition of 1-mM ATP. The addition of either 3-μM FCCP or 2-μM NH4Cl abolished the H+ gradient created by ATP hydrolysis.
NO measurement and localization
The NO was imaged using 4,5-diaminofluorescein diacetate (DAF-2 DA) with a fluorescence microscope. Root transverse sections from mature zones treated for 72 h were loaded with 10-μM DAF-2 DA in 10-mM HEPES-BTP buffer (pH 7.5) for 40 min, washed three times in fresh buffer and analyzed microscopically (488 nm/495–575 nm excitation/emission). The transverse root sections were ~ 5 μm and were created using a table microtome (LPC model, Rolemberg e Bhering Trading and Import, Belo Horizonte, Brazil). Images acquired from the light microscope (Zeiss Axioplan coupled with a Canon A640 digital camera) were analyzed using ImageJ software in the LR zone (~ 30 mm from the root–seed junction). Maize roots without DAF-2 DA addition were used as blank controls. The same camera settings were used, and the digital images were not processed further. The effects of the NO donor sodium nitroprusside (SNP, 200 μM) and the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO, 200 μM) on NO production were investigated. At least three samples were measured per treatment in three independent experiments.
N-Isopropyldecanamide 13C NMR (100 MHz) and 1H (400 MHz) H chemical shift (δ) spectral
2.12 t (J = 7.4)
0.88 t (J = 7.0)
4.09 sp (J = 6.6)
2′ and 3′
1.4 d (J = 6.6)
HA characterization by CP–MAS 13C and 15N NMR
Root dry mass, length and surface area
Root mitotic sites and lateral root emergence
The proliferation of mitotic sites in the root meristematic zones of 7- and 15-day-old plants treated with HA or N-isopropyldecanamide are shown in Fig. 6. The lower N-isopropyldecanamide (10−8 M) treatment promoted an increase in the number of mitotic sites and lateral root emergence, which were very similar to the effects of HA. The stimulative effects observed at 7 days were maintained at 15 days.
H+ pumping and ATP hydrolysis
PM vesicles isolated from maize roots treated for 7 day with 20 mg C L−1 HA or 10−4, 10−6 or 10−8 M N-isopropyldecanamide exhibited clear vanadate-sensitive stimulative effects (Fig. 7a) on the ATP-dependent proton gradient’s formation and ATPase activity (Fig. 7b). Both the initial rate of gradient formation and ATP hydrolysis were enhanced by fourfold and threefold in response to HA and N-isopropyldecanamide (10−6 and 10−8 M) treatments, respectively. Thus, the H+ pump may be involved in the alkamide-related stimulation of root growth, in a manner similar to that previously observed for HA.
N-Isopropyldecanamide, HA and SNP induced NO accumulation in maize root
Alkamides contain an acyl chain linked by an amide bond to an amine-containing head group. The nature of the alkyl amine group may vary, with butyl, isobutyl and propyl groups having been reported. The best studied alkamide is N-isobutyldecatrienamine, also named affinin . Here, we synthesized N-isopropyldecanamide and observed the presence of decanamide in the unbound fraction associated with HA aggregation (Figs. 1 and 8). The presence of small lipids in the humic fraction had been previously revealed through humeomics, the sequential chemical fractionation of humic matter from different sources [28, 29, 30, 31, 32] as well as their chemical conformations  and activities . However, here, for the first time, the bioactivities of HA were linked to the presence of alkamides. HA affect nutrient uptake through the synthesis and functionality of membrane proteins, especially proton pumps that increase the electrochemical proton gradient across the PM . Owing to their crucial roles in ion uptake and root growth, they can be used as biochemical markers of HA bioactivity . While the effects of alkamides on root growth are known [21, 22], their effects on PM H+-ATPase were not considered in previous reports.
The most common effects of HS on plant development are related to hormonal and the auxin-like activities . However, a new group of plant growth-regulating substances has an apparently auxin signaling-independent response . The influence of HS on different enzymes has been demonstrated . Here, the clear stimulation of root development by in vivo N-isopropyldecanamide and HA treatments was shown and correlated with an enhanced PM H+-ATPase activity in 7- and 15-day-old plants. Since auxin inhibitors could only partially impair HA bioactivity , it seems that the remaining HA effects could be related to alkamides. The 10−6- and 10−8-M N-isopropyldecanamide treatments, which enhanced root length and superficial area significantly, positively altered the PM H+-ATPase activity, as assessed by two- to threefold increases in ATP hydrolysis and ATP-dependent H+ transport compared with control plants. Root growth promotion by HA has been reported and can be explained, at least in part, by an enhancement in PM H+-ATPase activity. In this work, a 10−8-M N-isopropyldecanamide treatment had effects on the initial and steady-state H+ gradient rates that were very similar to those of HA.
The lateral root formation induced by HA is a well-studied NO-mediated process . The role of NO in the alterations induced by N-isobutyldecanamide during lateral root emergence in Arabidopsis was studied by Méndez-Bravo et al. . They observed a modulation in auxin-inducible gene expression and lateral root promotion through the interactions of alkamides with signals from jasmonic acid and NO. They concluded that N-isobutyldecanamide and its interacting signals with jasmonic acid and NO act downstream or independently of auxin-responsive gene expression to promote lateral root formation . In addition, López-Búcio et al.  showed that alkamides may belong to a class of endogenous signaling compounds that interact with the cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development. Changes in the expression of the cell division marker CycB1:uidA and the enhanced expression of the cytokinin-inducible marker ARR5:uidA occur both in roots and in shoots after plant exposure to alkamides. The presence of alkamides in the HA may contribute to the increased plant cell signaling and accelerated metabolism. The cellular energy balance could be altered as demonstrated by the increase in PM H+-ATPase activity induced by alkamides and HA.
We describe for the first time the presence of N-isopropyldecanamide in the unbound fraction of HA isolated from cattle manure vermicompost. A synthesized alkamide promoted maize root growth in a manner similar to that of HA. In addition, the effects of N-isopropyldecanamide on the PM H+-ATPase activity and NO accumulation in maize roots were shown. In this study, we provide evidence that alkamides enhance PM H+-ATPase activity and that the bioactivity levels of HA are not only a result of auxin-related effects, but also the presence of a mixture of plant growth regulatory substances.
LPC is grateful to FAPERJ and CNPq for the financial support to NUDIBA´s Lab.
DBZ carried out the plant experiments; CM wrote the first version of this paper; CRRM synthesized the N-isopropyldecanamide and confirmed its structure by spectroscopy methods; RNC found the N-isopropyldecanamide in the supramolecular arrangement of humic acids; RS did the humic acid characterization using CP/MAS NMR; LPC conceived the experiment and wrote the final version. All authors read and approved the final manuscript.
This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento de Pesquisa e Tecnologia (CNPq).
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The authors declare that they have no competing interests.
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