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Humic acids and Herbaspirillum seropedicae change the extracellular H+ flux and gene expression in maize roots seedlings



The use of plant-based biostimulants to increase crop yield and enhance protection has grown in popularity. We previously manufactured an inoculant combining humic acids with plant growth-promoting bacteria and observed a significant effect on crop yields. However, electrophysiology studies elucidating the mechanisms responsible for enhanced nutrient and water uptake are scarce and are generally restricted to in vitro models.

Materials and methods

In this study, a suspension of humic acids isolated from vermicompost along with Herbaspirillum seropedicae was applied to maize seedlings. We measured the root H+ flux with an ion-selective vibrating probe system. Furthermore, the transcription of plasma membrane H+ ATPase, aquaporin and high- or low-affinity nitrate transporters was measured.


Inoculation activated the extracellular H+ flux, thus changing the pH and membrane potential of maize root cells and altering the electrochemical potential generated by P–H+-ATPase at the biochemical and molecular level. The overexpression of aquaporins was also observed; however, nitrate transporters were repressed by the inoculants.


We demonstrate an increase in the H+ efflux in maize root seedlings inoculated with H. seropedicae and concomitant changes in membrane voltage. The inoculation of H. seropedicae in the presence of humic acids decreased the H+ flux and surface acidification without changes in the aquaporin transcription level. However, the H+ flux in root seedlings in inoculated plants (with or without humic acids) was larger in respect to control plants. These results support the increased water and nitrogen efficiency in plants inoculated with humic acids and H. seropedicae.


Humic substances (HS) comprise a major part of soil organic matter and influence soil processes and fluxes including soil fertility, water retention and the fate of xenobiotics [1]. HS can be used directly on plants in low concentrations to enhance plant growth, yield and nutrient uptake, thus constituting a category of plant biostimulants [2]. The effects of HS on plant growth and crop yield have been studied previously [3]. HS enhanced the efficiency of nutrient use, thus aiding in the assimilation of both macro- and microelements and inducing primary and secondary plant metabolism [3]. It was reported that humic acids (HA) can stimulate plant growth through the release of bioactive molecules with hormonal-like activity [4], thus directly affecting enzymatic activities of several metabolic pathways [2, 5, 6]. The effect of HS on plant nutrition may be mediated by the synthesis and functionality of membrane proteins, especially proton pumps that increase the electrochemical proton gradient across the plasma membrane (P) [7, 8]. The first report of proton pump stimulation by HS was provided by Maggioni et al. [9]. Due to its crucial role in ion uptake and root growth, this phenomenon has been well studied [10,11,12,13,14,15,16,17]. HA can increase the activity of plasma membrane (P) H+-ATPase (P–H+-ATPase) and vacuolar H+-ATPase, thus modulating the cellular electrical environment and proton pump activity that are used as biochemical markers of HA bioactivity [18].

Assuming that pH changes precede or reflect ongoing processes [19], pH could serve as a messenger where pH changes are needed for certain processes such as root growth [18]. The P–H+-ATPase could transcend its classical functions as an energy transducer system and assume a key role as a signal transducer [20]. The bioactive compounds in the HA supramolecular arrangement can be recognized by transmembrane receptor proteins [21, 22] or access the cytoplasm and thus modify the cytosolic pH (pHcit). Small variations in the pHcit play a critical role in several cellular processes, including the induction of enzymatic activities and ion transport [23]. Indeed, the activation of the P-type H+-ATPase increases the extracellular H+ flux, thus making the apoplast more acidic and providing an optimal medium for the activity of expansin proteins, which are fundamental for cell expansion [24, 25]. The activation of H+ flux is also crucial for energizing secondary transporters [8]. The enhancement of nutrient uptake, root growth promotion and absorption of water are among the main biostimulation effects of HS [26] and are responsible for the increase in crop yields in soils with low fertility [27, 28].

In highly weathered soils, low nitrogen is often cited as the main limiting factor in crop yield and results in the high cost of fertilizers. The use of plant growth-promoting bacteria (PGPB) is a sustainable alternative that enhances the efficiency of nutrient usage [28]. Biological nitrogen fixation and the solubilization of soil phosphorus are the main targets of commercial biostimulants that are manufactured using PGPB [29]. However, the success of PGPB in non-leguminous plants is substantially less than the results obtained in rhizobia–legume symbioses. In non-nodulating plant species, PGPB need to colonize epiphytic and endophytic tissues, and these processes are influenced by environmental stress and microbiological competition with native species.

The use of HA to introduce PGPB to crops has been documented [27, 30, 31]. Olivares et al. [32] reviewed the basic mechanisms and benefits of the combined application of HA and PGPB to several crop species. The suitability of HA as an adjuvant to bacterial foliar sprays was demonstrated for field-grown maize, where yields were 65% higher than untreated controls and 40% higher than maize fields treated with HA or bacteria [27]. Moreover, a boost in the metabolic profile of sugarcane leaves induced by HA and PGPB [33] with activation of the glycolysis pathway and enhanced TCA activity resulted in the accumulation of α-ketoglutaric acid, which is a central metabolite in glutamine and glutamic acid synthesis. The stimulation of TCA enzymes by HS was previously reported by Nardi et al. [34].

Regulation of the electrochemical environment at the cell/aqueous solution interface by HA and PGPB has not been well investigated. Numerous mechanism have been shown to function in the PGPB–host plant interaction, including the alleviation of abiotic stress, siderophore production, the production of 1-aminocyclopropane-1-carboxylate deaminase (ACC), phytohormone production, antifungal activity, the induction of systemic resistance, and interference with pathogen toxin production [35]. The effect of PGPB alone or in combination with HA on proton pump activity has been minimally studied despite the influence on nitrate and water uptake. The aim of this work was to evaluate H+ flux in root cells treated with HA and PGPB and the effects on transcription of genes encoding nitrate transporters and aquaporins.

Materials and methods

HA obtainment and its chemical derivatives

The HA used in this study was isolated from the vermicompost of filter cake, which is a typical product of the sugar industry in the north of Rio de Janeiro State, Brazil. HA was obtained according to the classical method of extraction, isolation and purification described in the web page of the International Humic Substances Society ( After freeze drying by lyophilization, the carbon content was analyzed by dry combustion (CHN analyzer Perkin Elmer series 2400, Norwalk, CT, USA). The chemical nature of HA was accessed by cross-polarization magic angle spinning (CP/MAS) 13C nuclear magnetic resonance (13C-NMR). The spectrum was acquired from the solid sample with a Bruker Avance 500 MHz (Bruker, Karlsruhe, Germany), equipped with a 4 mm wide bore MAS probe, operating at a 13C-resonating frequency of 75.47 MHz. The spectra were 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).

Obtainment of microbial inoculum

The bacterium used was Herbaspirillum seropedicae strain HRC54, which was originally isolated from sugarcane roots [36]. The pre-inoculum was prepared from a frozen bacterium stored at 15% glycerol. An aliquot of 50 µL was grown in 5 mL tube containing liquid nutrient broth (NB) medium for 36 h at 30 °C under orbital shaker at 150 rpm and checked under phase contrast microscopy for contamination and later by colony morphology in JNFb solid medium. A 200 mL Erlenmeyer flask containing 50 mL of NB liquid medium was inoculated with 100 µL of the pure culture pre-inoculum and was grown at the same condition reported above for 48 h. After that, the bacteria cells were harvested, centrifuged, washed, and the optical density adjusted to 1.8 at 460 nm (approximately 109 cells per mL).

Plant growth and treatment with humic materials and PGPB

Maize seeds (Zea mays L. var. UENF 506-11) were disinfected through immersion in a solution of NaClO (1.0%) for 30 min. Then, the seeds were washed and placed in distilled water for a period of 6 h. After, seeds were germinated in paper, in the dark, at 28 °C. Four days after germination, maize seedlings with approximately 0.5–0.7 cm were transferred to pots containing a minimal medium solution (2 mM CaCl2) and the treatments were: 0 (control), 2 mM C L−1 of the HA, Herbaspirillum seropedicae strain HRC54 produced at a concentration of 109 viable cells per mL, according to Döbereiner et al. [37] (bacteria) or the combination of HA and bacteria (HA + B). The pH of the solution was adjusted to 5.8–6.0. After 7 days in hydroponic culture, the seedlings were harvested and the analyses were performed. The minimal medium (2 mM CaCl2) was used to avoid any influence of the nutrients, which could act synergistically with the HA, stimulating root growth and seedlings’ metabolism [32].

Dry mass and root length

After 7 days in hydroponics culture, roots were dried in forced air circulation oven at a temperature of 50 °C for 48 h and weighed on an analytical balance. Root length was measured with a digital caliper. Data are presented as an average of four repetitions.

Extraction of total RNA

100–200 mg of fresh root tissues were homogenized with a mortar and pestle in the presence of liquid N2. The homogenate was transferred to new RNAse-free microcentrifuge tubes (1.5 mL) and the RNA was extracted using the mini-plant RNeasy Qiagen® kit (Germantown, USA).

Reverse transcription (RT) followed by polymerase chain reaction (PCR)

1–2 µg of total RNA was used for production of cDNAs. The synthesis was performed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). A PCR with a gradient temperature (59, 60 and 61 °C) was performed to confirm the specificity of the primers and the actual melting temperature. Electrophoresis in 2.0% agarose gel with TAE buffer was also performed for the confirmation of PCR products with the specific primers. Primers for the genes ubiquitin (UBI), aquaporin (PIP), low-affinity nitrate transporter (Nrt2.1), high-affinity nitrate transporter (Nrt1.1), PM-H+-ATPase (Mha1) were designed with Primer3 program and its characteristics were evaluated in Oligothech program and after a rigorous analysis, they were synthesized by IDT technology. Confirmation of primers specificity was obtained in a high-resolution gel, which gave single PCR products at the different temperatures tested and with the expected size. Specificity was also confirmed by melting curve performed in Step OneTMApllyedBiossystems®.

Real-time PCR (qRT-PCR)

For statistical validation of the experiments, two independent tests in the thermal cycler Step OneTMApllyedBiossystems®, with mRNA extracted from the independent experiments, were performed. cDNAs of each experiment were used in quadruplicate for each condition evaluated. The medium for the PCR was prepared as follows (final concentrations): 5 ρMof the forward primer (Additional file 1: Table S1), 5 ρM of the reverse primer (1 μL) (Additional file 1: Table S1), 7.5 µL of SYBR Green I component (ApllyedBiossystems®) and 0.5 µL of ultra-pure water. 10 µL of medium was added to an ELISA plate and 5 µL of cDNA was added. For cDNA dilution curve, the following concentrations were used: 0.2, 2, 20 and 200 ng of cDNA template at the control condition. The whole procedure was performed in a laminar flow using sterile materials that were RNAse free. After the addition of the reagents, the plate was sealed with adhesive and centrifuged gently. The protocol used for the experiment consisted of four steps: (i) program denaturation (10 min at 95 °C); (ii) amplification program and quantification repeated 45 times (10 s at 95 °C; 5 s at 61 °C—for both genes [reference and target]; 5 s at 72 °C with a single fluorescence acquisition mode); (iii) melting curve program (65–95 °C with a heating rate of 0.1 °C per s with continuum fluorescence acquisition) and (iv) cooling program to lower the temperature to 40 °C. Crossing points (CPs) were obtained and used in the subsequent calculations. CPs are defined as the point at which the fluorescence achieves significantly higher levels than non-specific fluorescence.

The relative mRNA expression of the interest genes and the endogenous control (ubiquitin [UBI]) (Additional file 1) were compared using a nonparametric pairwise fixed reallocation randomization test® as previously described [38]. The formula used to calculate the relative expression is as follows:

$$R = \frac{{\left( {E_{\text{target}} } \right)\Delta {\text{CP}}_{\text{target}} \left( {{\text{Control}}\, {\text{averarge}} - {\text{sample}}\, {\text{average}}} \right)}}{{\left( {E_{\text{ref}} } \right)\Delta {\text{CP}}_{\text{ref}} \left( {{\text{Control}}\,{\text{averarge}} - {\text{sample}}\,{\text{average}}} \right)}}$$

The equation represents the mathematical model used to calculate the ratio of relative expression in RT-qPCR. The ratio R of a target gene for a specific condition (sample or treatment) is presented versus the control condition compared to the reference gene (ref). Etarget is the efficiency of RT-qPCR of the reference gene transcript. ΔCPtarget is the difference between the CP of the target genes transcripts at the control condition and the sample or treatment condition. ΔCPref is the difference between the CP of the reference gene transcript at the control condition and the sample or treatment condition.

Measurements of H+ fluxes using the ion-selective vibrating probe system

Extracellular H+ fluxes were measured at the elongation zone of the main root of maize, treated or not with HA or PGPB, using an ion-selective vibrating probe technique [39,40,41]. Maize seedlings were placed in plastic Petri dishes (140 × 140 mm) filled with 20 mL of low salt-adapted nutrient medium composed by: 0.037 mM NaH2PO4, 1 mMKCl, 0.1 mM MgSO4, 0.5 mM Ca(NO3)2, 0.1 mL L−1 Clark micronutrients, 0.025 mL L−1 Fe-EDTA, pH 5.5. Ion-specific vibrating microelectrodes were produced as described by Feijó et al. [39]. Micropipettes were pulled from 1.5 mm borosilicate glass capillaries ( using the Puller Flaming Brown, Sutter P-98 (Sutter Instruments, Novato, CA) and treated with N,N-dimetiltrimetilsilamina (C5H15NSi, Fluka 41716). After silanization, they were backfilled with a 15–20 mm column of electrolyte (15 mM KCl and 40 mM KH2PO4, pH 6.0) and then frontloaded with a 20–25 µm column of H+-selective liquid exchange cocktail (Fluka, Milwaukee, WI, USA). An Ag/AgCl wire electrode holder (World Precision Instruments, Sarasota, FL, USA) was inserted into the back of the microelectrode and established electrical contact with the bathing solution. The ground electrode was a dry reference (DRIREF-2, World Precision Instruments) that was inserted into the sample bath. Signals were measured using an amplifier (, while the vibration and the electrode positioning were obtained through motors (stepper motors), allowing a three-dimensional movement. The control of the motors, data acquisition and its primary processing were adjusted through the ASET 2.0 software (Science Wares [East Falmouth, MA]— The calibration of the electrodes was conducted by measuring the potential (mV) recorded in three solutions containing the ion studied, with known concentrations: 0.1 mM, 1 mM and 10 mM, given that concentrations cover the conditions of the means used. The collection of data from the ion-selective vibrating probe, performed through the ASET software, provided the necessary information to calculate the ionic flow at a given point [x, y, z] of space, through Fick’s law (J = D (dc/dx)). The diffusion coefficient (D) is a standard value for each ion (in accordance with Handbook of Chemistry and Physics, Chemical Rubber Co.). The spatial difference (dx) resulted from the calculation of the distance between the two points that were made the measurements of concentrations to calculate the flow (15 μm). The concentration difference (cd) is a vector that varies during the test. At each point, the concentration can be calculated from the mV value recorded and the predetermined equation for the ionophore during the calibration process.

Statistical analysis

The experiment was arranged in randomized block designs with four biological replicates. The results were statistically analyzed by one-way ANOVA combined with Tukey`s test at P < 0.05, using the program GraphPad Prism 7.0.


Humic acid (HA) characteristics

The HA isolated from sugarcane filter cake alone or in conjunction with H. seropedicae exhibited low carbon (46%), high nitrogen (5.7%) and high oxygen content (45%) compared with HA from pedogenic sources. As expected, the chemical nature of HA was characterized by an increase in carbohydrate moieties as revealed by CPMAS 13C NMR spectrum (Additional file 2: Figure S1). The main signals present in the spectrum were a broad signal around 30 ppm due to CH3 and CH2 groups and two sharp peaks at 56 ppm and 72 ppm, which can be attributed to methoxy and O-alkyl groups, respectively. The broad resonance between 120 and 152 ppm is typical of aromatic and olefinic carbons, while the intense signal at 174 ppm reveals a large quantity of carboxyl groups compatible with high oxygen content as revealed by elemental composition analysis.

Dry mass and root length

There was no significant difference in the root dry mass among the treatments (Fig. 1a). However, root length was stimulated by HA + Bac when compared to control plants (Fig. 1b). There was no significant difference in root length for the remaining treatments.

Fig. 1
figure 1

a Dry mass and b length of maize root seedlings treated with 50 mg C L−1 humic acid (HA), Herbaspirillum seropedicae (Bac) or 50 mg C L−1 humic acid and H. seropedicae (HA + Bac) for 7 days in hydroponic culture. Data were analyzed by one-way ANOVA combined with Tukey’s test at P < 0.05 (n = 4)

Gene expression

The relative expression of Mha1 was sixfold higher (P < 0.05) for all treatments (HA, Bac and HA + Bac) with respect to the control (Fig. 2a). No statistical differences were observed among treatments (Fig. 2a). ZmPIP1 encodes aquaporin 1 protein and functions in water transport. The relative transcription of this gene was six-, eight- and ninefold (P < 0.05) higher in HA, Bac and Bac + HA treatments, respectively, when compared to the control (Fig. 2b). Repressed transcription of genes involved in NO3 transport was observed in all treatments (Fig. 3a, b), but was higher when HA or Bac were used singly than when the two treatments were combined (HA + Bac) (Fig. 3a, b).

Fig. 2
figure 2

Relative mRNA expression of the a P–H+-ATPase (Mha1) and b aquaporin (ZmPIP1) genes in maize roots treated or untreated with 50 mg C L−1 humic acid (HA), H. seropedicae (Bac) or 50 mg C L−1 humic acid and H. seropedicae (HA + Bac) for 7 days in hydroponic culture. mRNA levels were normalized in relation to the control gene (ubiquitin). Errors were calculated by the REST® program

Fig. 3
figure 3

Relative mRNA expression of the a low-affinity nitrate transporter (Nrt2.1) and b high-affinity nitrate transporter (Nrt1.1) genes in maize roots treated or untreated with 50 mg C L−1 humic acid (HA), H. seropedicae (Bac) or 50 mg C L−1 humic acid and H. seropedicae (HA + Bac) for 7 days in hydroponic culture. mRNA levels were normalized in relation to the control gene (ubiquitin). The error was calculated by the REST® program

Proton flux, root surface pH and voltage differences

The H+ flux at the elongation zone of maize roots was stimulated in all treatments when compared to the control; the highest flux was observed in the Bac treatment, followed by HA + Bac and HA only (Fig. 4a). An H+ influx of − 2.0 pmol was observed at the elongation zone of control plants (Fig. 4b). Roots treated with HA exhibited an H+ efflux of 3.5 pmol, and roots treated with Bac presented an even higher efflux of 25.0 pmol (Fig. 4a). In contrast, the combined treatment of HA + Bac provoked a reduction in the efflux of roots, with an average value of 12.0 pmol.

Fig. 4
figure 4

a H+ flux and b root surface pH at the elongation zones of Zea mays L. primary roots treated or untreated with 50 mg C L−1 humic acid (HA), H. seropedicae (Bac) or 50 mg C L−1 humic acid and H. seropedicae (HA + Bac) for 7 days in hydroponic culture. Data were analyzed by one-way ANOVA combined with Tukey`s test at P < 0.05 (n = 10)

Changes in root surface pH correlated with H+ flux measurements. Significant differences were observed in the Bac treatment with lower pH (4.8) values; no significant differences were detected in the pH values of other treatments relative to the control (Fig. 4b). All treatments had positive voltage values, whereas the control treatment presented positive and negative H+ voltage values (Fig. 5). Plants treated with Bac exhibited the highest H+ voltage increase (mean of 6.0 µV), followed by HA + Bac and HA, with mean values of 3.5 and 1.0 µV, respectively (Fig. 5).

Fig. 5
figure 5

Graphical representation of differences in H+ current (µV) at the elongation zones of Zea mays L. primary roots treated or untreated with 50 mg C L−1 humic acid (HA), H. seropedicae (Bac) or 50 mg C L−1 humic acid and H. seropedicae (HA + Bac) for 7 days in hydroponic culture. Orange lines represent reference values


Inoculants containing PGPB are important tools in overcoming problems associated with the excessive use of chemical fertilizers and pesticides [42]. In Brazil, the use of PGPB in non-leguminous plants remains restricted in comparison with soybeans. The co-inoculation of PGPB and HA resulted in increased yields for maize, sugarcane, pineapple, common bean and tomatoes [32]. The broad use of this low cost, environmentally friendly technology is partly dependent on understanding how the inoculants function in plant physiology. Here we describe the effect of H. seropedicae (Bac) and HA in H+ fluxes and membrane transporters.

The relative expression of Mha1 in maize seedlings treated with HA, Bac or HA + Bac showed increased expression and was up to sixfold higher than control plants. Façanha et al. [43] previously showed that solutions containing HA increased the number of P–H+-ATPases isolated from mono- and dicotyledonous roots. P–H+-ATPases play a central role in plant metabolism, because their activity generates an electrochemical gradient in cell membranes; these gradients are required for the activation of ion translocation, which is fundamental for the absorption of macro- and micronutrients [44]. In addition, the activity of P–H+-ATPases results in acidification of the apoplast and promotes increased cell wall plasticity, which facilitates growth and division of plant cells [45]. Canellas et al. [46] verified that the regulation of P–H+-ATPase synthesis by HAs isolated from vermicompost is typical of functional auxinic clusters that were detected in their structure. HS isolated from vermicompost regulates the expression of Mha1 and Mha2, which are involved in the synthesis of P–H+-ATPase in a manner similar to auxins [47] and are responsible for the induction of lateral roots and the synthesis, activation and expression of P–H+-ATPases [48].

Water channel proteins known as the MIP (major intrinsic protein) family have been identified in most living organisms. In plants, MIP was first identified in bean and represented approximately 2% of total proteins extracted from cotyledons [49]. A water channel function was first demonstrated in plants for a protein isolated from Arabidopsis thaliana, opening new perspectives in understanding mechanisms that regulate the transmembrane flow of water [50]. In this work, all treatments (HA, Bac or HA + Bac) promoted increased expression of the aquaporin gene, ZmPIP1. Furthermore, we demonstrated the contribution of PIP1 and PIP2 in water transport within roots, especially during conditions of water deficit [51, 52]. A prior work demonstrated that the co-inoculation of HA and PGPB enhanced water stress recovery, and inoculated sugarcane and beans exhibited a larger water content in leaves than untreated plants [53, 54]. The function of PIP2 in the osmotic balance of A. thaliana root xylem cells was previously demonstrated by the analysis of T-DNA insertion mutagenesis [55]. Furthermore, an increase in aquaporin gene expression during N deficiency was observed by Liu et al. [56] and during salt stress when barley was inoculated with the nitrogen-fixing bacteria, Azospirillum brasilense [57]. The effect of HA in the differential expression of aquaporins in rice was previously described by Garcia et al. [58, 59].

The two main components directly involved in the uptake of NO3 are P–H+-ATPases and NO3 transporters. These components need to be coordinated with the availability of NO3 in the medium for more efficient absorption. The transcriptional response of plants to the differential availability of NO3 may be a key mechanism for survival in nutrient-poor environments; this could occur by differentially regulating specific genes for nutrient absorption, such as NO3 transporters and P–H+-ATPase [60, 61]. We confirmed the transcriptional repression of NO3 transport genes in all treatments (HA, Bac or HA + Bac). Increased expression of these genes was observed only in response to NO3 fertilization [47]; this demonstrated that low MW humic fractions indirectly stimulate NO3 uptake, possibly by increasing levels of Mha1 and Mha2 and inducing H+ fluxes in the rhizosphere.

We observed increased H+ flux in the elongation zone of maize roots treated with HA, Bac and HA + Bac. The increased H+ flux at the elongation zone of roots inoculated with H. seropedicae is likely due to the activation of P-type H+-ATPases in response to auxins secreted by the bacteria. Previous reports suggest that many of the benefits provided by H. seropedicae are linked to auxin production [62]. Auxin secretion by bacteria can result in increased root volume, higher numbers of lateral roots and a larger capacity for nutrient uptake, thus resulting in greater promotion of plant growth [27, 63, 64]. Increased amino acid production, which may be attributed to the induction of H+-ATPases already present in the cell [65, 66], indicate a form of post-transcriptional regulation.

Previously, an increase in H+ flux was reported for seedling roots treated with HA that was isolated from vermicompost [67]; this was associated with higher hydrolytic activity and H+ transport of P–H+-ATPases, which then promoted a higher emergence of lateral roots in rice seedlings. Along with the increased of H+ flux in plants treated with HA and HA + Bac, we predict an increase in the acidification of roots. This is likely because the P–H+-ATPase has a fundamental role in plant metabolism due to the activation of secondary transporters, acidification of the apoplast and nutrient absorption [68, 69]. Fluctuations in H+ efflux have been described with biochemical and molecular changes involving the P–H+-ATPases, thus indicating that the bioactivity of HA impacts these enzymes in the plants [7, 26, 70]. The activity induced by HA is regulated partially by auxin, other plant hormones, various compounds, and functional groups in HA [4]. In this study, we show that the use of H. seropedicae singly or in combination with HA modified the root seedling H+ flux and membrane voltage similarly but with different levels of magnitude; this is logical since H. seropedicae reportedly induced the synthesis of auxin-like compounds and changed the hormonal balance in plants [36].


The use of HA, Bac or HA + Bac altered the membrane potential of maize root cells, changing the electrochemical potential generated by P–H+-ATPase at both biochemical and molecular levels. We also observed changes in the expression of water transporters, thus revealing the physiological role of the combined inoculation of HA + Bac. The increase of H+ efflux and cellular water content is pivotal in efforts to enhance nutrient and water efficiency. Drought conditions are predicted to increase with climate change [71], along with problems due to the high cost and environmental impact of nitrogen fertilizers. The inoculation of plants with HA + Bac induces physiological changes that can help plants sustain the impact of reduced nitrogen and drought stress.



Humic acids


Cell suspension of Herbaspirillum seropedicae

HA + Bac:

Humic acids and Herbaspirillum seropedicae


Humic substances


Major intrinsic proteins






Low-affinity nitrate transporter


High-affinity nitrate transporter



NO3 :



Plant growth-promoting bacteria


Plasma membrane proton ATPase


  1. Piccolo A. The nature of sol organic matter and innovative soil management to fight global changes and maintain agricultural productivity. In: Piccolo A, editor. Carbon sequestration in Agricultural soils: a multidisciplinary approach to innovative methods. Heidelberg: Springer; 2012. p. 1–20.

    Chapter  Google Scholar 

  2. Roomi S, Masi A, Conselvan GB, Trevisan S, Quaggiotti S, Pivato M, Arrigoni G, Yasmin T, Carletti P. Protein profiling of arabidopsis roots treated with humic substances: insights into the metabolic and interactome networks. Front Plant Sci. 2018;9:1812.

    Article  Google Scholar 

  3. Nardi S, Ertani A, Ornella F. Soil-root cross-talking: the role of humic substances. J Plant Nutr Soil Sci. 2017;180:5–13.

    Article  CAS  Google Scholar 

  4. Nardi S, Pizzeghello D, Muscolo A, Vianello A. Physiological effects of humic substances in higher plants. Soil Biol Biochem. 2002;34:1527–37.

    Article  CAS  Google Scholar 

  5. Conselvan GB, Fuentes D, Merchant A, Peggion C, Francioso O, Carletti P. Effects of humic substances and indole-3-acetic acid on Arabidopsis sugar and amino acid metabolic profile. Plant Soil. 2018;426:17–32.

    Article  CAS  Google Scholar 

  6. Olaetxea M, Hita D, García AC, Fuentes M, Baigorri R, Mora V, Garnica M, Urrutia O, Erro J, Angel M, Zamarreño AM, Berbara RL, Garcia-Mina JM. Hypothetical framework integrating the main mechanisms involved in the promoting action of rhizospheric humic substances on plant root- and shoot growth. Appl Soil Ecol. 2018;123:521–37.

    Article  Google Scholar 

  7. Nardi S, Carletti P, Pizzeghello D, Muscolo A. Biological activities of humic substances. In: Senesi N, Xing B, Huang PM, editors. Biophysico-chemical processes involving natural nonliving organic matter in environmental systems, vol. 2., Part 1: fundamentals and impact of mineral-organic biota interactions on the formation, transformation, turnover, and storage of natural nonliving organic matter (NOM)Hoboken: Wiley; 2009. p. 305–39.

    Chapter  Google Scholar 

  8. Palmgren MG. Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake. Ann Rev Plant Physiol. 2001;52:817–45.

    Article  CAS  Google Scholar 

  9. Maggioni A, Varanini Z, Nardi S, Pinton R. Action of soil humic matter on plant roots: stimulation of ion uptake and effects on (Mg2+ + K+) ATPase activity. Sci Total Environ. 1987;62:355–63.

    Article  CAS  Google Scholar 

  10. Nardi S, Concheri G, Dell’Agnola G, Scrimin P. Nitrate uptake and ATPase activity in oat seedlings in the presence of two humic fractions. Soil Biol Biochem. 1991;23:833–6.

    Article  CAS  Google Scholar 

  11. Pinton R, Varanini Z, Vizzotto G, Maggioni A. Soil humic substances affect transport properties of tonoplast vesicles isolated from oat roots. Plant Soil. 1992;142:203–10.

    Article  CAS  Google Scholar 

  12. Varanini Z, Pinton R, De Biasi MG, Astolfi S, Maggioni A. Low molecular weight humic substances stimulated H+-ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots. Plant Soil. 1993;153:61–9.

    Article  CAS  Google Scholar 

  13. Pinton R, Cesco S, Santi S, Varanini Z. Soil humic substances stimulate proton release by intact oat seedling roots. J Plant Nutr. 1997;20:857–69.

    Article  CAS  Google Scholar 

  14. Pinton R, Cesco S, Iacoletti G, Astolfi S, Varanini Z. Modulation of NO3 uptake by water-extractable humic substances: involvement of root plasma membrane H+-ATPase. Plant Soil. 1999;215:155–61.

    Article  CAS  Google Scholar 

  15. Canellas LP, Olivares FL, Okorokova-Façanha AL, Façanha AR. Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots. Plant Physiol. 2002;130:1951–7.

    Article  CAS  Google Scholar 

  16. Quaggiotti S, Ruperti B, Pizzeghello D, Francioso O, Tugnoli V, Nardi S. Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). J Exp Bot. 2004;55:803–13.

    Article  CAS  Google Scholar 

  17. Zandonadi DB, Canellas LP, Façanha AR. Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation. Planta. 2007;225:1583–95.

    Article  CAS  Google Scholar 

  18. Zandonadi DB, Santos MP, Caixeta LS, Marinho EB, Peres LEP, Façanha AR. Plant proton pumps as markers of biostimulant action. Sci Agric. 2016;73:24–8.

    Article  CAS  Google Scholar 

  19. Felle HH. pH: signal and messenger in plant cells. Plant Biol. 2001;3:577–91.

    Article  CAS  Google Scholar 

  20. Zandonadi DB, Santos MP, Dobbss LB, Olivares FL, Canellas LP, Binzel ML, Okorokova-Facanha AL, Facanha AR. Nitric oxide mediates humic acids-induced root development and plasma membrane H+-ATPase activation. Planta. 2010;231:1025–36.

    Article  CAS  Google Scholar 

  21. Hottz-Rima JA, Martim SA, Dobbss LB, Evaristo JAM, Retamal CA, Façanha AR, Canellas LP. Adição de ácido cítrico potencializa a ação de ácidos húmicos e altera o perfil protéico da membrana plasmática em raízes de milho. Ci Rural. 2011;41:614–20.

    Article  Google Scholar 

  22. Carletti P, Masi A, Spolaore B, De Laureto PP, De Zorzi M, Turetta L, Ferretti M, Nardi S. Protein expression changes in maize roots in response to humic substances. J Chem Ecol. 2008;34(6):804.

    Article  CAS  Google Scholar 

  23. Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev. 2010;110:2709–28.

    Article  CAS  Google Scholar 

  24. Rayle DL, Cleland RE. The acid growth theory of auxin-induced cell elongation is alive and well. Plant Physiol. 1992;99:1271–4.

    Article  CAS  Google Scholar 

  25. Barbez E, Dünser K, Gaidora A, Lendl T, Busch W. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana. Proc Natl Acad Sci. 2017;114:E4884–93.

    Article  CAS  Google Scholar 

  26. Canellas LP, Olivares FL. Physiological responses to humic substances as plant growth promoter. Chem Biol Technol Agr. 2014;1:3.

    Article  Google Scholar 

  27. Canellas LP, Martínez-Balmori D, Médici LO, Aguiar NO, Campostrini E, Rosa RC, Façanha A, Olivares FL. A combination of humic substances and Herbaspirillum seropedicae inoculation enhances the growth of maize (Zea mays L). Plant Soil. 2013;366:119–32.

    Article  CAS  Google Scholar 

  28. Adesemoye AO, Torbert HA, Kloepper JW. Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microb Ecol. 2009;58:921–9.

    Article  CAS  Google Scholar 

  29. Owen D, Williams AP, Griffith GW, Withers PJA. Use of commercial bioinoculants to increase agricultural production through improved phosphorus acquisition. Appl Soil Ecol. 2015;86:41–54.

    Article  Google Scholar 

  30. Olivares FL, Aguiar NO, Rosa RCC, Canellas LP. Substrate biofortification in combination with foliar sprays of plant growth promoting bacteria and humic substances boosts production of organic tomatoes. Sci Hortic. 2015;183:100–8.

    Article  Google Scholar 

  31. Silva SF, Olivares FL, Canellas LP. The biostimulant manufactured using diazotrophic endophytic bacteria and humates is effective to increase sugarcane yield. Chem Biol Technol Agric. 2017;4:24.

    Article  Google Scholar 

  32. Olivares FL, Busato JG, Paula AM, Lima LS, Aguiar NO, Canellas LP. Plant growth promoting bacteria and humic substances: crop promotion and mechanisms of action. Chem Biol Technol Agric. 2017;4(1):30.

    Article  Google Scholar 

  33. Aguiar NO, Olivares FL, Novotny EH, Canellas LP. Changes in metabolic profiling of sugarcane leaves induced by endophytic diazotrophic bacteria and humic acids. Peer J. 2018;6:e5445.

    Article  Google Scholar 

  34. Nardi S, Muscolo A, Vaccaro S, Baiano S, Spaccini R, Piccolo A. Relationship between molecular characteristics of soil humic fractions and glycolytic pathway and Krebs cycle in maize seedlings. Soil Biol Biochem. 2007;39:3138–46.

    Article  CAS  Google Scholar 

  35. Bhattacharyya PN, Jha DK. Plant growth promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol. 2012;28:1327–50.

    Article  CAS  Google Scholar 

  36. Olivares FL, Baldani VLD, Reis VM, Baldani JI, Döbereiner J. Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems and leaves predominantly of Gramineae. Biol Fertil Soil. 1996;21:197–200.

    Article  Google Scholar 

  37. Dobereiner J, Baldani VLD, Baldani JI. Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Brasília: Embrapa-SPI; 1995. p. 60.

    Google Scholar 

  38. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30:1–10.

    Article  Google Scholar 

  39. Feijó JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK. Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J Cell Biol. 1999;144:483–96.

    Article  Google Scholar 

  40. Ramos AC, Façanha AR, Feijó JA. Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi. New Phytol. 2008;178:177–88.

    Article  CAS  Google Scholar 

  41. Ramos AC, Lima PT, Dias PN, Kasuya MCM, Feijó JA. A pH signaling mechanism involved in the spatial distribution of calcium and anion fluxes in ectomycorrhizal roots. New Phytol. 2009;181:448–62.

    Article  CAS  Google Scholar 

  42. Vassilev N, Vassileva M, Lopez A, Martos V, Reyes A, Maksimovic I, Löbermann B, Malusà E. Unexploited potential of some biotechnological techniques for biofertilizer production and formulation. Appl Microbiol Biotechnol. 2015;99:4983–96.

    Article  CAS  Google Scholar 

  43. Façanha AR, Façanha ALO, Olivares FL, Guridi F, Santos GA, Velloso ACX, Rumjanek VM, Brasil F, Schripsema J, Braz-filho R, Oliveira MA, Canellas LP. Bioatividade de ácidos húmicos: efeito sobre o desenvolvimento radicular e sobre a bomba de prótons da membrana plasmática. Pesq Agropec Bras. 2002;37:1301–10.

    Article  Google Scholar 

  44. Sze H. H+-Translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol. 1985;36:175–208.

    Article  CAS  Google Scholar 

  45. Cosgrove DJ. Loosening of plant cells walls by expansins. Nature. 2000;407:321–6.

    Article  CAS  Google Scholar 

  46. Canellas LP, Teixeira Junior LRL, Dobbss LB, Silva CA, Medici LO, Zandonadi DB, Façanha AR. Humic acids cross interactions with root and organic acids. Ann Appl Biol. 2008;153:157–66.

    CAS  Google Scholar 

  47. Quaggiotti S, Ruperti B, Pizzeghello D, Francioso O, Tugnoli V, Nardi S. Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). J Exp Bot. 2004;55:803–13.

    Article  CAS  Google Scholar 

  48. Frías I, Caldeira MT, Pérez-Castineira JR, Navarro-Avinó JP, Culianez-Maciá FA, Kuppinger O, Stransky H, Pagés M, Hager A. Serrano R) A major isoform of the maize plasma membrane H+-ATPase: characterization and induction by auxin in coleoptiles. Plant Cell. 1996;8:1533–44.

    PubMed  PubMed Central  Google Scholar 

  49. Johnson KD, Herman EM, Chrispeels MJ. An abundant, highly conserved tonoplast protein in seeds. Plant Physiol. 1989;91:1006–13.

    Article  CAS  Google Scholar 

  50. Maurel C, Reizer J, Schroeder JI, Chrispeels MJ. The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes. EMBO J. 1993;12:2241–7.

    Article  CAS  Google Scholar 

  51. Kaldenhoff R, Grote K, Zhu J-J, Zimmermann U. Significance of plasmalemma aquaporins for water-transport in Arabidopsis thaliana. Plant J. 1998;14:121–8.

    Article  CAS  Google Scholar 

  52. Siefritz F, Otto B, Bienert GP, Van Der Krol A, Kaldenhoff R. The plasma membrane aquaporin NtAQP1 is a key component of the leaf unfolding mechanism in tobacco. Plant J. 2004;37:147–55.

    Article  CAS  Google Scholar 

  53. Aguiar NO, Medici LO, Olivares FL, Dobbss LB, TorresNetto A, Silva SF, Novotny EH, Canellas LP. Metabolic profile and antioxidant responses during drought stress recovery in sugarcane treated with humic acids and endophytic diazotrophic bacteria. Ann Appl Biol. 2016;168:203–13.

    Article  CAS  Google Scholar 

  54. da Piedade Melo A, Olivares FL, Médici LO, Torres-Neto A, Dobbss LB, Canellas LP. Mixed rhizobia and Herbaspirillum seropedicae inoculations with humic acid-like substances improve water-stress recovery in common beans. Chem Biol Technol Agric. 2017;4:6.

    Article  Google Scholar 

  55. Javot H, Lauvergeat V, Santoni V, Martin-Laurent F, Guçlu J, Vinh J, Heyes J, Franck KI, Schaffner AR, Bouchez D, Maurel C. Role of a single aquaporin isoform in root water uptake. Plant Cell. 2003;15:509–22.

    Article  CAS  Google Scholar 

  56. Liu L-H, Ludewig U, Gassert B, Frommer WB, von Wirén N. Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis. Plant Physiol. 2003;133:1220–8.

    Article  CAS  Google Scholar 

  57. Zawoznik MS, Ameneiros M, Benavides MP, Vázquez S, Groppa MD. Response to saline stress and aquaporin expression in Azospirillum-inoculated barley seedlings. App Microbiol Biotech. 2011;90:1389–97.

    Article  CAS  Google Scholar 

  58. García AC, Santos LA, Izquierdo FG, Sperandio MVL, Castro RN, Berbara RLL. Vermicompost humic acids as na ecological pathway to protect rice plant against oxidative stress. Ecol Eng. 2012;47:203–8.

    Article  Google Scholar 

  59. García AC, Santos LA, Izquierdo FG, Rumjanek VM, Castro RN, Santos FS, Souza LGA, Berbara RLL. Potentialities of vermicompost humic acid to alleviate water stress in rice plants (Oryza sativa L.). J Geochem Expl. 2014;136:48–54.

    Article  Google Scholar 

  60. Araki R, Hasegawa H. Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction. Breed Sci. 2006;56:295–302.

    Article  CAS  Google Scholar 

  61. Santi S, Locci G, Monte R, Pinton R, Varanini Z. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transport and plasma membrane H+-ATPase isoforms. J Exp Bot. 2003;54:1851–64.

    Article  CAS  Google Scholar 

  62. Bastián F, Cohen A, Piccoli P, Luna V, Bottini R, Baraldi R. Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media. Plant Growth Reg. 1998;24:7–11.

    Article  Google Scholar 

  63. Baldani VD, Baldani JI, Döbereiner J. Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. Biol Fert Soils. 2000;30:485–91.

    Article  Google Scholar 

  64. Alves GC, Videira SS, Urquiaga S. Reis VM Differential plant growth promotion and nitrogen fixation in two genotypes of maize by several Herbaspirillum inoculants. Plant Soil. 2015;387:307–21.

    Article  CAS  Google Scholar 

  65. Hager A. Role of the plasma membrane H+-ATPase in auxin-induced elongation growth: historical and new aspects. J Plant Res. 2003;116:483–505.

    Article  CAS  Google Scholar 

  66. Takahashi K, Hayashi KI, Kinoshita T. Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis thaliana. Plant Physiol. 2012;159:632–41.

    Article  CAS  Google Scholar 

  67. Ramos AC, Olivares FL, Silva LS, Aguiar NO, Canellas LP. Humic matter elicits proton and calcium fluxes and signalling dependent on Ca2+-dependent protein kinase (CDPK) at early stages of lateral plant root development. Chem Biol Technol Agric. 2015;1:1–12.

    Google Scholar 

  68. Sze H, Li X, Palmgren MG. Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. Plant Cell. 1999;11:677–89.

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Morsomme P, Boutry M. The plant plasma membrane H+-ATPase: structure, function and regulation. Biochim Biophys Acta. 2000;1465:1–16.

    Article  CAS  Google Scholar 

  70. Dobbss LB, Canellas LP, Olivares FL, Aguiar NO, Peres LEP, Spaccini R, Piccolo A. Bioactivity of chemically transformed humic matter from vermicompost on plant root growth. J Agric Food Chem. 2010;58:3681–8.

    Article  CAS  Google Scholar 

  71. Reganold JP, Wachter JM. Organic agriculture in the twenty-first century. Nat Plants. 2016;2:15221.

    Article  Google Scholar 

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Authors’ contributions

IGA carried out the experiments and wrote the first version of this paper; FLO and LPC contributed to the experimental idea. ACR and AAB were responsible for the ion-selective vibrating probe system analysis. All authors read and approved the final manuscript.


We would like to acknowledge Dr. Evenilton Costa Pessoa for the contribution on the real-time PCR analysis. We are particularly grateful to the associate editor and anonymous reviewer that made a number of helpful comments.

Competing interests

The authors declare that they have no competing interests.

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This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), 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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Correspondence to Luciano Pasqualoto Canellas.

Additional files

Additional file 1: Table S1.

Primer sequence of the genes analyzed. Ubiquitin (Ubi), aquaporin (Pip), low-affinity nitrate transporter (Nrt2.1), high-affinity nitrate transporter (Nrt1.1), P–H+-ATPase (Mha1). F = Forward, R = Reverse.

Additional file 2: Figure S1.

CP-MAS 13C NMR spectrum of humic acids used in the experiments.

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de Azevedo, I.G., Olivares, F.L., Ramos, A.C. et al. Humic acids and Herbaspirillum seropedicae change the extracellular H+ flux and gene expression in maize roots seedlings. Chem. Biol. Technol. Agric. 6, 8 (2019).

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