206
10.1002/jpln.201500228
J. Plant Nutr. Soil Sci. 2016, 179, 206–214
Phosphorus speciation and high-affinity transporters are influenced by
humic substances
Keiji Jindo1,2, Tatiane Sanches Soares1, Lázaro Eustáquio Pereira Peres3, Inga Golçalvez Azevedo1,
Natália Oliveira Aguiar1, Pierluigi Mazzei4, Riccardo Spaccini4, Alessandro Piccolo4, Fábio Lopes Olivares1,
and Luciano Pasqualoto Canellas1*
1
Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Núcleo de Desenvolvimento de Insumos Biológicos para Agricultura Av.
Alberto Lamego, 2000, CEP 28013–602 Campos dos Goytacazes, Rio de Janeiro, Brazil
2 Institute of Industrial Science, the University of Tokyo, 3–8–1 Komaba, Meguro-ku, Tokyo 153–8902, Japan
3 Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’ (ESALQ), Universidade de São Paulo (USP),
Piracicaba, Brazil
4 Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare, CERMANU, Università di Napoli Federico II, Portici, Italy
Abstract
Phosphorus (P) is a limiting factor for plant growth, especially in highly weathered tropical soils.
Plants have several mechanisms to overcome low P availability in soil, such as humic substances, that reduce phosphate (Pi) adsorption on oxide surfaces and enhance soil P availability.
However, the direct influence of humic substances on Pi transporters in root cells or the distribution of P species in leaves remains unclear. Tomato seedlings were grown in a sand–vermiculite
mixture with low or high P concentrations (10 or 100 mg kg–1 KH2PO4, respectively) and humic
acids (0 or 48 mg C L–1) isolated from vermicompost. Plant responses were evaluated in the fifth
week by measuring root and shoot weights and P concentration, and differential expression in
the roots of the high-affinity Pi transporter genes LePT1 and LePT2. In addition, the distribution
of P species in the leaves was assessed using 31P-NMR. Humic acids increased the root biomass and changed the distribution of P species in the leaves. Inorganic phosphate was the major compound in plants supplied with a high P concentration, whereas in plants supplied with a
low P concentration, Pi was only identified in plants not treated with humic acids. Glycerophosphodiester and phosphorylcholine accumulated in plants treated with humic acid, indicating a
modified metabolic pathway for economical P consumption at low P concentrations. High transcript accumulation of LePT2 was observed in roots treated with humic acids at both P concentrations. Our results show that humic substances are strategically involved in plant adaptation to
P availability.
Key words: humic acids / phosphate transporter / vermicompost
Accepted February 04, 2016
1 Introduction
Phosphorus (P) plays a vital role in plant growth and development as it is a component of many cell constituents, such as
nucleotides and phospholipids, and it is essential for energy
conservation and metabolic regulation (Cheng et al., 2011).
However, P availability is critical for plant productivity because the forms of inorganic P (Pi) absorbed by plants are
poorly soluble in the soil solution (López-Bucio et al., 2002).
Despite the large amounts of total P in tropical soils, where Al
and Fe are dominant, the bioavailable forms of P are minimal
due to Pi retention by oxides and clay minerals and P immobilization into organic forms (Hufnagel et al., 2014). Furthermore, approximately half of the world’s agricultural land comprises soils of low P content (Lynch, 2011).
There are various mechanisms of plant adaptation to low P
availability (Ticconi et al., 2004; Wissuwa et al., 2005; Arnaud
et al., 2014). They include elongation of root systems to
enhance the exploited soil volume (Ticconi et al., 2004), exudation of organic anions and protons to mobilize P from insoluble forms (Jones and Darrah, 1994), and secretion of
phosphatases by roots and microorganisms (Wasaki et al.,
2009). Humic substances influence all of these mechanisms.
Humic matter consists of a large variety of natural organic
molecules originating from dead biological material, especially plant tissues affected by decomposition and microbial
activity (Orsi, 2014). These organic materials form supramolecular associations of heterogeneous and relatively small
molecules (< 1000 Da) that are held together by weak linkages such as hydrogen bonds and hydrophobic bonds
(Piccolo, 2002). Such weak linkages are easily broken by the
organic anions exuded by plants, thereby liberating a plethora
of small humic molecules that can access cell receptors and
influence plant growth (Canellas and Olivares, 2014).
* Correspondence: L. P. Canellas; e-mail: [email protected]
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J. Plant Nutr. Soil Sci. 2016, 179, 206–214
Rose et al. (2014) reviewed the biological effects of humic
substances and calculated a net 20% increase in plant growth
when plants were exposed to humic matter. Humic substances induce H+-ATPase activity in the plasma membrane
(Canellas et al., 2002), control organic anion exudation (Canellas et al., 2008; Puglisi et al., 2009), influence microbial
communities in the rhizosphere (Puglisi et al., 2013), and
modulate the extracellular activity of acid phosphatases (Garcı́a et al., 1995). Erro et al. (2012) showed that humic acids increase Pi solubility and prevent P immobilization in soil; but
otherwise, there are few investigations of the direct influence
of humic acids on plant P transporters and speciation.
In environments with low P availability, plants modify their
metabolic processes for efficient P use by inducing changes
in the lipid composition of plasma membranes, by increasing
Pi transporter activity in root tissues, by building new temporal
starch and anthocyanins, and by differentiating gene expression (Ticconi et al., 2004; Wissuwa et al., 2005; Arnaud et al.,
2014; Hufnagel et al., 2014). These responses are a combination of general stress-related responses and P-specific
signaling cascades. Consequently, a diverse range of subproducts and side effects of plant metabolism occur in plant
tissues due to P starvation. For example, phospholipids are
replaced with chloroplast sulfolipids, carbohydrates are translocated from shoots to roots, amino acid concentrations (e.g.,
glutamine and asparagine) are increased, and levels of phosphorylated intermediates in the leaves (e.g., glucose-6-P,
fructose-6-P, inositol-6-P, and glycerol-3-P) are reduced,
which results in changes in P speciation (Huang et al., 2008).
P speciation and HAT affected by humic substances 207
The humic acids (HA) were extracted from 10 g of vermicompost with 100 mL of a 0.1 M NaOH solution under N2 atmosphere. This procedure was repeated several times until the
supernatant became colorless. The extracts were united and
centrifuged at 5000 g for 15 min. The supernatant was then
acidified with 6 M HCl to pH 2.0 and kept at 4°C for 12 h. The
precipitated HA was separated by centrifugation from the
soluble fulvic acid that remained in the supernatant. The HA
was purified by treating it three times with 10 mL of a dilute
HF (0.3 M) + HCl (0.1 M) solution. After centrifugation at
4000 g for 15 min, the sample was washed repeatedly with
water, dialyzed against deionized water using a 1 kDa cutoff
membrane, and lyophilized to maintain stability until use.
2.2 Humic acids characterization
The HA elemental composition was determined using a CHN
Perkin Elmer autoanalyzer (Perkin Elmer series 2400, Norwalk, CT, USA). The O content was calculated from the difference (i.e., O% = 100 – C% – H% – N%) on an ash-free basis,
and the elemental composition was: C = 46%, N = 5.7%;
H = 3.0%; O = 45%.
With the assumption that humic substances contribute to
plant performance at low available P concentrations and affect plant metabolism, we hypothesized that humic acids
change both P speciation in the leaves and the expression of
high-affinity Pi transporters in root cells, and thereby contribute to plant adaptation and growth at low available P concentrations in the soil.
Cross-polarization magic-angle spinning (CPMAS) 13C nuclear magnetic resonance (13C-NMR) spectra were acquired
from the solid samples with a Bruker Avance 500 MHz
(Bruker, Karlsruhe, Germany), equipped with a 4 mm widebore 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 to 46 ppm (alkyl C, mainly
CH2 and CH3 sp3 carbons), 46 to 65 ppm (methoxy and N alkyl C from OCH3, C–N, and complex aliphatic carbons), 65 to
90 ppm (O-alkyl C, such as alcohols and ethers), 90 to
108 ppm (anomeric carbons in carbohydrate-like structures),
108 to 145 ppm (phenolic carbons), 145 to 160 ppm (aromatic
and olefinic sp2 carbons), 160 to 185 ppm (carboxyl, amides,
and esters), and 185 to 225 ppm (carbonyls; Spaccini and
Piccolo, 2009).
2 Material and methods
2.3 Tomato seedling experiment
2.1 Vermicompost production and humic acids
isolation
Vermicompost was prepared using sugarcane filter cake and
ground sugarcane from a commercial factory. The sugarcane
juice was first cleaned with sulfur and then with calcium to
promote colloid flocculation. The colorless cleaned juice was
evaporated, and then vacuum- filtered to separate it from a
stacked solid remaining on the filter, which is called filter
cake. The filter cake was placed in a concrete cylinder
(100 cm internal diameter) with a 150 L capacity, and the humidity was kept at 65–70% after mixing by weekly additions
of water. Two cylinders (two replicates) were prepared per
treatment. After approximately 1 month, earthworms (Eisenia
foetida) were introduced at a ratio of 5 kg of worms per m3 of
organic residue. At the end of the transformation process
(4 months after the distribution of the last organic residues),
the worms were removed by placing a pile of fresh organic
residue in a corner of the container.
ª 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Tomato (Solanum lycopersicum L. cv. Micro-Tom) seeds
were surface-sterilized by soaking in 0.5% NaClO for 30 min.
After rinsing and soaking in distilled water for 6 h, the seeds
were planted into pots containing a 2.5 L mixture of vermiculite and sand (2 : 1). The seeds were supplied with nutrient
solution according to the method of Clark (1982): 0.6 mM of
MgSO4; 0.9 mM of NH4NO3; 0.5 mM of KCl; 1.3 mM of KNO3;
2.53 mM of Ca(NO3)2; 13.3 mM of H3BO4; 7 mM of MnCl2;
2 mM of ZnSO4; 2 mM of CuSO4; 0.086 mM (NH4)6Mo7O2;
75 mM Fe-EDTA. Two different concentrations of P (10 and
100 mg kg–1 KH2PO4) and lyophilized humic acids
(4 mM C L–1) were added to the nutrient solution to provide
samples treated with HA (10+HA and 100+HA). Nutrient
solutions without HA were considered as control treatments
(10 and 100 mg kg–1). The concentration of HA (4 mM C L–1)
was based on a previously determined optimum concentration for tomato growth (Canellas et al., 2011). The four treatments were replicated six times in a completely randomized
statistical design. Five weeks after germination, the dry
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Jindo, Peres, Azevedo, Aguiar et al.
weights of roots and shoots were measured in three replicates. The root and shoot samples were frozen using liquid
N2 and stored at –60°C. For P quantification, the frozen root
and shoot samples were finely ground, treated with H2SO4,
placed into a block digester, and heated to 200°C. After cooling, the samples were treated with 0.5 mL of 30% H2O2 and
heated again to 200°C. Heating and H2O2 additions were repeated continually until the solution was clear. Thus, transparent solution of the sample, taken from the supernatant, was
diluted with distilled water and analyzed for P with ascorbic
acid using a spectrophotometer (Shimadzu, Tokyo, Japan).
Details of the procedures are described in Watanabe and
Olsen (1965). The root and shoot dry weights and P analysis
were reported as mean values. All results were statistically
analyzed using analysis of variance and Tukey’s honest significant difference post-hoc test.
2.4
31P-NMR
spectra of leaf extracts
Leaf extracts were obtained according Bligny et al. (1990).
Tomato leaves (5 g, wet weight) were quickly frozen at liquid
nitrogen temperature to avoid ATP destruction and then finely
ground with 1mL of 70% (w/v) perchloric acid. The frozen
powder was then placed at –10°C and thawed. The thick suspension thus obtained was centrifuged at 10,000 g for 10 min
to remove particulate matter. The supernatant was neutralized with 2 M KHCO3 to about pH 6.0 and centrifuged at
10,000 g for 10 min to remove precipitated KClO4. Next, 2 mL
of 120 mM trans-1,2-diaminocyclohexane-N,N,N¢,N¢-tetraacetic acid (CDTA) was added. The resulting supernatant was
lyophilized and stored at –80°C. For the NMR measurement
100 mg of this freeze-dried material, containing 120 mM
CDTA, was dissolved in 2 mL of 40 mM HEPES buffer
(pH 7.8) containing 10% of D2O and analyzed with 31P-NMR
on a Bruker Avance 500 MHz (Bruker, Karlsruhe, Germany)
spectrometer. NMR spectra were obtained by applying the following parameters: pulse program HPDEC (high power decoupling); 13,000 Hz rotor spin rate; 10 s recycle time; 30 ms
acquisition time; 6000 scans. Fourier transformation was performed with 4000 data points and an exponential apodization
with 50 Hz of line broadening. Signals and peaks were assigned to individual P chemical compounds or functional
groups in accordance with established data. A capillary filled
with methylene diphosphonic acid (0.36 mM) resonating at
16.5 ppm was used as an external chemical-shift reference.
2.5 Gene transcription of high-affinity Pi
transporter genes LePHT1 and LePHT2
in tomato
To evaluate the differential gene transcription of high-affinity
Pi transporters in the tomato plant roots, RNA was extracted
using the RNeasy Plant Mini kit (74904, Qiagen, Germantown, USA) following the manufacturer’s instructions. Subsamples of frozen root (100 mg) were finely ground in liquid
N2 using a mortar and pestle and placed into an RNase-free
2 mL microcentrifuge tube. RLT buffer (450 mL) was added
and the sample was vortexed. The supernatant was transferred to a new microcentrifuge tube, and absolute ethanol
(0.5 mL) was added to the cleared lysate and mixed immedi-
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J. Plant Nutr. Soil Sci. 2016, 179, 206–214
ately by pipetting. RW1 buffer (700 mL) was added to the
RNeasy spin column and centrifuged at 10,397 g for 15 s to
wash the spin column membrane; the flow-through was discarded. RPE buffer (500 mL) was added to the RNeasy spin
column and centrifuged at 10,397 g for 15 s to wash the spin
column membrane; this was repeated once. The RNeasy
spin column was placed in a new 1.5 mL collection tube and
RNase-free water (40 mL) was added to the spin column
membrane and centrifuged at 10,397 g for 1 min to elute the
RNA. The cDNAs were synthesized using the High Capacity
cDNA Reverse Transcription (RT) Kit (Applied Biosystem,
ThermoFisher, USA), which involved 1 · RT buffer, 1 · dNTP
Mix, 1 · RT Random Primers, 1 mL of MultiScribe Reverse
Transcriptase, 1 mL of RNase Inhibitor, 2 mg of RNA, and Nuclease-free H2O to complete the reaction. The thermal cycler
conditions were 25°C for 10 min, 37°C for 120 min, and 85°C
for 5 min. Semiquantitative PCR was performed using the
specific primers designed from the Lpt1 and Lpt2 sequence:
Lpt1fw (5¢-AAC-GAA-GGG-GAA-GAG-GAA-AC–3¢) and
Lpt1rv (5¢-GCA-TTG-TAG-TGT-ATA-CTA-ACT-G-3¢); Lpt2fw
(5-GGA-AGC-ATC-ACA-AGA-AAC-TAT-A-3¢) and Lpt2rv
(5¢-CTT-ACA-CAA-TAC-AAA-GAA-AAC-TG-3¢). Tomato actin
specific primers were used for DNA amplification control:
Tomactfw (5¢-TTC-CGT-TGC-CCA-GAG-GTC-CT-3¢) and
Tomactrv (5¢-TCG-CCC-TTT-GAA-ATC-CAC-ATC-3¢). PCR
conditions were: 29 cycles of 95°C for 50 s, 60°C for 50 s,
72°C for 50 s, and 72°C for 5 min. Gene transcriptions were
analyzed using ImageJ software (NIH, Bethesda, Maryland).
ANOVA (P < 5%) and Tukey’s Multiple Comparison Test
(*P < 5%, **P < 1%, ***P < 0.1%, ****P < 0.01% and ns: not significant) were used for statistical analysis. Statistical analysis
was calculated using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA).
3 Results
3.1 Characterization of the humic acids
The molecular composition of the HA was determined using
13C-NMR spectroscopy. The NMR spectrum (Fig. 1) shows
broad signals around 30 ppm due to CH3 and CH2 groups
originating from plant waxes and lipids. The peaks at 56 ppm
and 72 ppm were assigned to methoxy and O-alkyl groups
from cellulose, hemicellulose, and lignin structures (Spaccini
and Piccolo, 2009). The broad resonance between 120 and
152 ppm represents aromatic and olefinic carbons, while the
intense signal at 174 ppm reveals a large content of carboxyl
groups.
3.2 Plant biomass weight and P content
The influence of HA on plant biomass is reported in Fig. 2.
The shoot dry weight increased with HA treatments at both
P levels (Fig. 2A) but the root weight was not affected by HA.
At low P concentration control plants showed a greater
root : shoot ratio than the HA-treated plants. The P concentration in leaves and roots was related to the P concentration in
the plant nutrient solution. The HA treatment with low P concentration did not change the P concentration in the plant
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P speciation and HAT affected by humic substances 209
Dry weight / g
J. Plant Nutr. Soil Sci. 2016, 179, 206–214
Chemical shift / ppm
3.3
31P-NMR
analysis of the leaf extract
Phosphorus speciation of the leaf extracts obtained using
31P-NMR spectroscopy (Fig. 4) shows different signals and
intensities according to plant treatments with or without HA.
The peak assigned to inorganic Pi (H2PO4 ) at 2.7–2.9 ppm
was the most prominent NMR signal for both P levels. At the
low P concentration Pi was only identified in plants not treated
with HA (Fig. 4A) and they had a lower intensity than the
0.36 mM standard methylene diphosphonic acid. The P signal
for glucose-6-P at 4.0 ppm occurred with high intensity in control plants at the low P concentration (Fig 4A). However, in
plants treated with HA two additional high-intensity signals
appeared at 3.1 and 1.1 ppm, which could be assigned to
glyceryl phosphoryl glycose and phosphorylcholine, respectively (Fig. 4B). At the high P concentration, the leaf extracts
showed a signal at 4.2 ppm, attributed to fructose-6-P, which
was most intense for plants grown without HA (Fig. 4C). The
signal for organic P, attributed to glucose-6-P, was shifted to
3.95 ppm in leaf extracts from HA-treated plants (Fig. 4D).
3.4 Differential gene transcription
The results for the differential gene transcription of two highaffinity P transporters are shown in Fig. 5. Major differences
in transcription intensity were detected in LePT2, a P transporter gene reported to be intensively expressed in roots
(Hufnagel et al., 2014). In contrast, LePT1, known as a typical
gene having a ubiquitous transcription profile, did not significantly differ between the treatments. The transcript accumulation of LePT2 was strongly intensified in the HA treatment
with low P concentration with respect to the control, thus reflecting an increase in transporter synthesis and promoted P
uptake. As expected, at the high P concentration, transcription of LePT2 was inhibited, whereas HA addition surprisingly
induced gene transcription.
ª 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Dry weight / g
leaves (Fig. 3A), but both HA treatments increased the P concentration in the roots by about 20% (Fig. 3B).
Dry weight / g
Figure 1: CPMAS NMR spectrum of humic acids.
Figure 2: Dry weights of shoots (A), root (B), and root : shoot ratios
(C) of tomato seedlings treated with two different P concentrations
(10 and 100 mg kg–1 KH2PO4) with or without humic acids. The bars
represent the – standard deviations of the means (n = 10). The
Tukey’s test (P < 5%) for comparison of the means are reported as
minor letters above each bar.
4 Discussion
A very important issue of our time is to increase the production of food, fiber, and biofuel without increasing the environmental impact. After N, the nutrient most frequently deficient
in tropical soils is P, and the use of high quantities of P fertilizer can lead to waste and inefficient use. Moreover, as ferti-
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P content / g kg
–1
P content / g kg
–1
stances can act as plant growth regulators that
induce growth and development. In addition to
our results (Fig. 1), several other studies have
shown that humic substances increase plant
growth. For example, Rose et al. (2014) calculated that a 20% average increase in roots and
shoots biomass was induced by different types
and doses of humic substances in various
types of plants. Different research groups using
different approaches have demonstrated the
effects of humic substances on nutrient uptake
and stimulation of primary and secondary metabolism (Chen et al., 2004; Nardi et al., 2009;
Trevisan et al., 2011). Moreover, the main reported effect of humic substances on plants is
a change in root growth, including enhanced
lateral root emergence and root hair proliferation (Nardi et al., 2009). According to the acid
growth theory (Rayle and Cleland, 1992), H+
pumping lowers the pH of cell walls, activates
pH-sensitive enzymes and proteins associated
with the wall, and initiates cell-wall loosening
and extension growth. This mechanism is induced by auxins. Previous reports have demonstrated that humic substances can stimulate
the H+-ATPase of PM vesicles isolated from
the roots of several plants (Nardi et al., 1991;
Varanini et al., 1993; Pinton et al., 1999; Nardi
et al., 2002). Canellas et al. (2002) showed a
clear stimulation of the vanadate-sensitive
ATPase activity by HA and an enhanced
synthesis of this enzyme. Quaggiotti et al.
(2004) reported that overexpression of the
major isoform of maize, PM H+-ATPase (Mha
2), was induced by humic substances. The active H+ secretion through the ATPase drives
the uptake of ions, which maintains the internal
turgor pressure at a constant value during cell
Figure 3: Phosphorus concentrations of the leaves (A), and roots (B) of tomato seedelongation. The active H+/Pi symporter could
lings treated with two different P concentrations (10 and 100 mg kg–1 KH2PO4) with or
without humic acids. The bars represent the – standard deviations of the means
be activated, thereby enhancing the P concen(n = 6). The Tukey’s test (P < 5%) for comparison of the means are reported as minor
tration in plants treated with HA (Fig. 3). Auxin
letters above each bar.
or auxin-like activity is also implicated in posttranscriptional regulation of high-affinity P
transporters (Schachtman and Shin, 2007). The HA used in
lizer becomes more expensive it will be increasingly important
this work have high auxin activities as demonstrated previto grow crops more efficiently in environments with lower nuously using mutant tomato seedlings with gene reporters for
trient inputs (Good et al., 2004). Plant strategies to acquire P
auxin activity; e.g., DR5::GUS (Canellas et al., 2011). Thereare oriented around enhancing soil exploration and mobilizing
fore, posttranscriptional regulation of LePT2 transporter
poorly available P pools in the rhizosphere (Lynch, 2011).
genes by HA is possible. This is supported by the well-known
Humic substances are involved in these two strategies as
result that P deprivation induces changes in root developthey induce lateral root emergence and root hairs (Nardi
ment, which is a mechanism for increasing P acquisition uset al., 2002), and enhance H+ and organic anion exudation
ing upstream regulators, such as the hormones auxin and
(Canellas et al., 2002; 2008; Puglisi et al., 2009). Here, for
ethylene, to change root morphology or architecture
the first time we describe two other strategies: the induction of
(Schachtman and Shin, 2007).
differential expression of high-affinity P transporters and the
modification of P species in leaves (Figs. 4 and 5).
Another mechanism for efficient P use under deficient conditions, besides secondary transport energization by the
At low P supply, high-affinity Pi transporters were induced by
ATPase system and induction of differential expression of
humic substances as demonstrated by semiquantitative PCR
high-affinity Pi transporters, is metabolic change induced by
analysis (Fig. 5). It is well known that several environmental
Pi deprivation. Mineral deficiencies usually affect the roots
factors can modulate differential expression of transporters,
but the main factor was clearly low Pi availability. Humic subfirst and then transmit signals to the leaves (Wilkinson and
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Chemical shift / ppm
Chemical shift / ppm
P speciation and HAT affected by humic substances 211
Chemical shift / ppm
Chemical shift / ppm
Figure 4: 31P-NMR spectra of tomato leaf extracts treated with two different P concentrations of the nutrient solution. (A) 10 mg kg–1 KH2PO4;
(B) 10 mg kg–1 KH2PO4 plus humic acids; (C) 100 mg kg–1 KH2PO4; (D)100 mg kg–1 KH2PO4 plus humic acids. Methylene diphosphonic acid
(0.36 mM) was used as an external chemical-shift reference at 16.5 ppm.
Davies, 2002). When plants are deprived of P, scavenging
systems are activated to recover P from lipids and nucleic
acids. As a result, lipid composition shifts from phospholipids
to galactolipids and sulfolipids, ribonucleases recover P from
nucleic acids, acid phosphatase activity increases, and P
transporter genes are induced (Ticconi and Abel, 2004). Using 31P-NMR, we demonstrated that HA change the P speciation in leaves (Fig. 4). Humic acids partially influence the upregulation of compounds involved in metabolic pathways of
plant tissues and change the root : shoot distribution of mineral nutrients (Mora et al., 2010; Hu et al., 2014).
In our study, despite using the same ratio of extract : solvent
in all sample preparations, we found a less intense NMR signal for H2PO4 in leaves from the low P concentration treatment than in the external chemical-shift reference (Fig. 5), but
HA-treated plants manifested two large peaks of other phosphorylated compounds. The high mobility and the assimilation
of these organic P compounds suggest that a more plastic response to P deprivation occurred with the HA treatments
compared to the controls. The presence of glycerophospho-
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diester and phosphorylcholine were previously reported to be
involved with the turnover, metabolism, and degradation of
phospholipid and glycerol of P-deprived plants (Cheng et al.,
2011; Vauclare et al., 2013). These changes in P distribution
could be attributed to a modified metabolic pathway for economizing P consumption in plants and maintaining higher P
concentrations in plant cells (Fig. 3). The glycerophosphodiester degradation pathway is considered a source of inorganic P under P deprivation and the accumulation of these
compounds is often shown in some physiological situations
involving membrane turnover or degradation (Cheng et al.,
2011). Phosphorylcholine is a component of the plant phospholipid network that is transiently upregulated to replace
phospholipids with galactolipids and sulfolipids in plant membranes in response to P deprivation, as well as in response to
salt stress (Kocourková et al., 2011). The identification of
phosphorylcholine only in plants treated with HA (Fig. 4) suggests that HA modulates the plant response to P deprivation.
To increase the P uptake capacity at low P concentrations,
plants synthesize more P transporter proteins in the plasma
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Figure 5: Transcription of phosphate transporter genes LePT1 and LePT2 in tomato roots after treatment with
10 mg kg–1 KH2PO4 (–P), 100 mg kg–1 KH2PO4 (+P), 10 mg kg–1 KH2PO4 + humic acid (–P HA) and
100 mg kg–1 KH2PO4 + humic acid (+P HA). ANOVA (P < 5%) and Tukey’s Multiple Comparison Test (*P < 5%,
**P < 1%, ***P < 0.1%, ****P < 0.01%, and ns: not significant). Statistical analysis was calculated using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Error bars correspond to the
standard errors of the means of three experiments.
membrane of root epidermal cells (Muchhal and Raghothama, 1999). LePT2, a member of the transporter PHT gene
family, has been documented as being expressed exclusively
in P-deprived roots (Chen et al., 2014). In our study, a greater
intensity of LePT2 transcript accumulation was found in
HA-treated roots supplied with low and high P concentrations
(Fig. 5). Since P transporters utilize the H+ gradient to drive
the cotransport process (Raghothama and Karthikeyan,
2005), enhancement of electrochemical gradient by activation
of the H+-ATPase plasma membrane by HA treatment could
support P transport even at low nutrient concentration. The
expression of LePT2 remained high with respect to control
plants at high P concentrations showing that HA may modulate the high-affinity Pi transporter independently of the P concentration of the solution. This unprecedented result may
have implications for soil-fertility management. The presence
of HA at high P concentration can modify the plant sensing
mechanism for nutrients and induce transcription of high-affinity Pi transporter LePT2 (Fig. 5).
Humic acids increased root and shoot dry weights, changed
the distribution of P species in leaves, enhanced organic
forms of P readily available at low P concentrations, and
induced concomitantly the synthesis of high-affinity Pi transporters in root cells. This unveils an adaptive mechanism in
response to low P availability. In addition, stimulation of high
affinity P transport occurred at high P concentrations, suggesting that HA can modulate P independently of the supplied
P concentration. The possibility of plant P toxicity after overexpression of P transporters at high external P supply cannot
be excluded.
Acknowledgments
This work was supported by the CAPES (‘‘Atração de Jovens
Talentos’’ Program-BJT). Additional funds were provided by
ª 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado
do Rio de Janeiro (FAPERJ), International Foundation of Science (IFS), and National Institute of Science and Technology
(INCT) for biological nitrogen fixation.
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