Influence of humic acid molecular fractions on growth and development of soybean seedlings under salt stress

Abstract

In the present study, the influence of humic acid (HA) molecular fractions (HA < 30 kDa and HA > 30 kDa) on enhancing the tolerance of seedlings of soybean [Glycine max (L.) Merr.] Progres and Nawiko cultivars to salt stress (50 mM NaCl) was investigated. HA were extracted from mountain fen soil and then were separated into two molecular fractions by membrane filtration and characterized by diffusion coefficient (Dapp), electrolytic conductivity (κ) and electrophoretic mobility (Ue). The following biometric parameters of tested plants were determined: total leaf area, height of plants, fresh and dry mass of the over ground part and roots as well as length of shoots cells and length of stomas. The chlorophyll content in ground tissue as well as the macro and microelements content in tested plants also were determined. The results showed that the κ, Ue and Dapp for HA > 30 kDa were lower than these for HA < 30 kDa. Adding NaCl caused increase κ and decrease Dapp and Ue. The salt stress caused a major decrease in biometric parameters in tested plants. HA > 30 kDa reduced the uptake of macro and microelements in the soybean Progres cultivar. In soybean Nawiko cultivar, it caused significant uptake of Fe and Zn. Soybean cultivars showed strong reaction to salt stress. HA molecular fractions reduced or eliminated the influence of the salt stress. However, HA > 30 kDa was more effective than HA < 30 kDa, due to its properties.

Introduction

One of the main abiotic stress factors limiting the growth of plants is soil salinity. More than 20 million hectares of land throughout the Europe are salt affected, this problem occurs mainly in Hungary, Romania, Greece, Italy and on the Iberian Peninsula. In the countries of northern Europe, when the salt is the most widely used de-icing agent for road maintenance, salinization occurs locally (Daliakopoulos et al. 2016).

Salt stress has various effects on plant physiological processes, e.g. it disturbs ion balance and contaminates with ions of Na+ and Cl (Starck et al. 1995; Chinnusamy et al. 2005), as well as changes in plant growth, mineral distribution, membrane instability and permeability, and decreases efficiency of photosynthesis (Munns 2002; Mansour and Salama 2004; Sudhir and Murthy 2004).

The salt tolerance of plants to soil salinity is not a permanent property of species. It depends on the growth stage, salt type and concentration, stress duration, environment conditions and other factors (Starck et al. 1995). Majority of crops are typical glycophites of minor tolerance to salt stress. Soybean [Glycine max (L.) Merr.] is a leguminous plant of major economic use. Soybean can be cultivated in a light moderate saline soil due to its moderate salt tolerance (Grieve et al. 2003).

It has been known for many years that humic substances (HS) are a very important component of soil influencing its physical, chemical and biological properties, in particular stability of soil aggregates, buffer properties, sorption of hydrophobic organic substances, transport, bio-absorption and complexation of metals in the environment. HS provide energy to soil microorganisms, have positive influence on soil moisture level, improve soil structure and make soil more fertile by releasing nutrients from soil minerals (Rosa et al. 2005; Ouni et al. 2014).

Humic substances influence plant growth both directly and indirectly, and in a positive and negative manner (Nardi et al. 2002). Studies, based on biometric factors, showed that HS enhance root, leaf and shoot growth but also stimulate the germination of various crop species (Piccolo et al. 1993; Calvo et al. 2014). Humic substances influence on root structure and growth dynamics, which leads to increased root size and density. Changes caused by HS also stimulate the H+-ATPase activity in cell membrane suggesting, that HS play an important role in its biochemical pathways (Canellas and Olivares 2014). The HS influence several metabolic processes, such as photosynthesis, respiration, nucleic acid synthesis and ion uptake (Nardi et al. 2002). For example, the humic substances influence the production of RNA, which is essential for many biochemical processes in the cell (Trevisan et al. 2010). Most HS bind tightly to plant cell walls and can be uptaken by roots whereas some of them can be transferred to the shoots (Nardi et al. 2009). Trevisan et al. (2010) reviewed the signaling events that influenced the physiological effects of HS on plant metabolism. As described above, humic acids have auxin-like effects on plants, and this primary effect was cited as the main biological factor accounting for the diverse beneficial effects on plants.

HS can be used as one of ways of counteracting negative consequences of stress factors (Kulikova et al. 2005; Çimrin et al. 2010; Aydin et al. 2012; Calvo et al. 2014; Ouni et al. 2014; Khalesro et al. 2015). Kulikova et al. (2005) drew attention to the fact that HS might show anti-stress effects under different abiotic stress conditions (unfavorable temperature, pH, salinity…etc). Studies have focused on using HS against salt stress are limited (Çimrin et al. 2010; Aydin et al. 2012; Khalesro et al. 2015). The activity of HS is depending on their structural characteristics (Berbara and Garcia 2014). Influence of humic acids (HA) on the growth of plants depends on a number of factors, e.g. their origin, concentration, molecular mass, application method and specie and development phase of a plant (Nardi et al. 2002).

Despite numerous reports published recently to elucidate the mechanisms underlying physiological effects of HS, the data reported are quite contradictory. The main reason is the complexity of the HS structure.

In soil prone to salinity (NaCl) and reduced content of organic matter, large quantity of sodium is absorbed by the humus complex, and simultaneously other elements are displaced, especially Ca. This makes the soil toxic to plants (Tchiadje 2007). The most effective method to improve plant growth in saline soil is to provide organic matter to reduce concentration of Na in soil, ion replacement and pH, probably due to the fact that together with HS elements such as Ca, Mg and K are introduced to the environment. The elements anchored in cation exchange HS centers and reduce adsorption of Na, which in turn promotes ions being washed out. Finally, it reduces salinity as it lower content of Na that is replaced by univalent K from a humus complex (Lakhdar et al. 2009). More information about HA counteracting negative environmental plant stress factors can be expected from current broad research.

Therefore, the aim of the present study was to assess the influence of molecular fractions of humic acids on reducing negative effects of salt stress in soybean of Progres and Nawiko cultivars.

Materials and methods

Analysis of physical and chemical properties of humic acid suspensions used in hydroponic production of soybean seedlings.

Mountain fen soil samples were collected from a surface layer from the Babia Góra National Park in the Polish Carpathians (N 49°35′45″–E 19°30′21″).

IHSS recommended method was used to extraction of humic acids (Swift 1996). Purified HA were separated into two molecular fractions by filtration on Amicon 8400 and Millipore filters with cut-off point 30 kDa.

Particle size distribution [hydrodynamic diameter (dh) range 0.6–6000 nm] was determined for HA suspensions in Michaelis buffer of pH 5.59, with and without NaCl at the beginning (0 h) and at the end of the experiment (after 196 h). The investigations were performed using the Dynamic Light Scattering (DLS) method (ZetaSizer Nano ZS apparatus; Malvern Instruments Ltd., UK) (ISO 13321 1996; Chu and Liu 2000; Pecora 2000; Kaszuba et al. 2008). Measurements were made at 20 °C in three series (each measurement consisted of 12 sub-measurements). During the hydroponic experiment, the mobility of humic acid molecules in the solution may determine their accessibility for plant roots, results are presented as diffusion coefficient (Dapp) distribution by intensity of scattered light. Additionally, electrolytic conductivity (κ) was determined for solutions tested (conductometry) and electrophoretic mobility (Ue) using the Laser Doppler Electrophoresis (LDE). Measurements of those parameters were made simultaneously in HA samples using ZetaSizer Nano ZS (Malvern Instruments Ltd., UK) (Mayinger 1994). Three series of measurements were applied (12 sub-measurements each) at 20 °C.

Plant growth conditions and treatments

The experiments were conducted in controlled conditions, using the Hoagland solution as a growth medium for seedlings of soybean [G. max (L.) Merr.] Progres and Nawiko cultivars.

Soybean seeds (100 pcs) were washed three times with distilled water and placed on trays with roasted silica sand. Moisture level was maintained at 50% of weighted water capacity. After 4–5 days, BBCH 10 growth phase (acc. to Munger et al. 2001) soybean were moved to germination apparatus of 0.08 × 0.012 × 0.036 m (height × width × length) and volume of 2.5 L made of white foamed PCV.

The experiment used water cultures with Hoagland growth medium of pH 5.59. Soybean seedlings and Hoagland medium were placed in miniphytotrone at controlled temperature and lighting [PPFD 350 μmol (photons) m−2 s−1, temperature of 25 °C, photoperiod of 16 h/8 h (day/night)]. To ensure proper oxygen supply the growth medium was aerated. After 7 days of the growth, NaCl was added to do Hoagland medium to ensure concentration of NaCl at 50 mM. One of two molecular HA fractions (<30 or >30 kDa) was added to the solution, concentration of which ensures carbon content in the HA solution of CHA = 0.005 g dm−3. pH of the medium has not changed after adding those fractions to the solution. The experiment consisted of four variations: control (growth medium Hoagland), NaCl (50 mM) + Hoagland growth medium, NaCl (50 mM) + HA <30 kDa + Hoagland growth medium, NaCl (50 mM) + HA > 30 kDa + Hoagland growth medium. After 7 days of growth in such conditions, soybean seedlings were taken for further tests.

Measurement of growth attributes

The content of chlorophyll was measured using SPAD-502 (Minolta CO. Ltd., Japan). The meter is used for non-invasive measurement of chlorophyll content in the ground tissue using the optic method. Results are expressed in SPAD, value of which is proportional to the content of chlorophyll in an examined area (6 mm2) of a leaf (Monje and Bugbee 1992).

Winfolia (Regent Instrument Inc., Canada) was used to determine the total leaf area of Progres and Nawiko cultivars.

The height of plants was measured, as well as fresh and dry mass of the over ground part and roots of Nawiko and Progres soybean seedlings. Dry mass was determined using drying in a dryer for 12 h at 105 °C.

Microbiometric measurements (electron microscopic examination)

Microbiometric measurements were performed based on microscope photographs made using an electron scanning with Quanta 200 by Fei. Photographs were made with magnification of 200, 300 and 1000 times at 1 kV difference in potential. Biological material consisted of parts of soybean tissue sampled directly before placing them in the measurement chamber. Parts of shoots and roots were cut lengthwise with a scalpel. Then, fresh samples were placed in a measurement chamber of a microscope and air pumped out. The number and length of stomas were determined based on pictured of the bottom part of leaves. The length of stomas was determined using microscope software, and the number was then converted into the number of stomas per leaf area.

Ion measurements

The content of four microelements (added to medium), including Cu, Mn, Zn, and Fe as well as Mg, Ca, K, and Na in the dry mass of the over ground parts of soybean seedlings. Over ground parts of soybean seedlings were dried to obtain dry mass at 35 °C, and then ground and mineralized in the mixture of concentrated HNO3 and HClO4. All markings were repeated in three series. After mineralizing, samples were subjected to the Atomic Absorption Spectrometry (AAS).

Statistical analyses

Data from measurements of physiological parameters underwent statistical analysis using Statistica 12.0. Variation analysis was used to compare mean values as a basis for separating homogenous groups using Tukey’s test at statistical the 0.05 significance level.

Multiple variance analysis was performed (ANOVA) (p < 0.05) to determine the influence of HA fractions, salinity and storage time on electrolytic conductivity and electrophoretic mobility. Within specific fractions of acids, a bi-factor analysis and post-hoc HSD Tukey’s test (Statistica v. 12.0, StatSoft Poland) were performed.

Results

Analysis of physical and chemical properties of humic acid suspensions

The properties of HA, which was a component of medium used for the soybean growth in hydroponics, changed with salinity and the duration of an experiment.

Figure 1 presents the distribution of the HA diffusion coefficient against intensity of light scattered in HA < 30 kDa suspension.

Fig. 1
figure1

Distribution of HA < 30 kDa diffusion coefficient at various salinity levels a directly after preparation of solutions, b after 196 h of storage

The fresh samples without electrolyte showed two overlapping peaks at the distribution of the diffusion coefficient. Eighty percent of the area was occupied by a peak with maximum value at the diffusion coefficient equal 1.02 µm2 s−1, which corresponds to particles of hydrodynamic diameter 404 ± 72 nm. The maximum of the second peak occurred at the diffusion coefficient 3.97 µm2 s−1. The peak corresponded to particles of dh 114 ± 15 nm. Once NaCl was added blunt peak appeared (98% of area) at the diffusion coefficient 1.18 µm2 s−1 for particles of 340 ± 95 nm and small peak (2% of area) at Dapp = 0.07 µm2 s−1 for particles of dh 5560 nm.

After 196 h, the distribution of the diffusion coefficient without electrolyte showed three peaks. It indicated the presence of particles with Dapp equal to 3.53 µm2 s−1 at hydrodynamic diameter 128 ± 12 nm (12% of area), 0.45 µm2 s−1 at hydrodynamic diameter 994 ± 135 nm (82% of area) and 0.08 µm2 s−1 for dh 5480 ± 118 nm (6% of area). Similarly, three peaks were found in the distribution of diffusion coefficient in the system with NaCl. The three peaks corresponded to three HA sub-fractions of Dapp = 1.55 µm2 s−1—for dh = 259 ± 30 nm (7% of area), 0.23 µm2 s−1 for dh = 1726 ± 310 nm (88% of area) and 0.08 µm2 s−1 for dh = 5256 ± 229 nm (5% of area).

Distributions of the diffusion coefficient against light intensity for HA fractions >30 kDa are presented in Fig. 2.

Fig. 2
figure2

Distributions of diffusion coefficient for HA > 30 kDa and various salt concentration a directly after preparation of suspensions, b after 196 h of storage

In the case of a fresh samples of HA fractions >30 kDa without NaCl, wide blunt peak at 1.33 µm2 s−1 corresponding to dh = 340 ± 130 nm prevailed at the distribution of the diffusion coefficient (92% of area). There was also a peak of Dapp = 0.09 µm2 s−1 which corresponded to dh = 4650 ± 410 nm (8% of area). With the presence of sodium salt two wide overlapping peaks were recorded, respectively at 1.54 µm2 s−1 (58% of area) for dh = 260 ± 70 nm and 0.22 µm2 s−1 (42% of area) for dh = 1790 ± 550 nm. After 196 h into the experiment, in a system without salt, the distribution of the diffusion coefficient had three peaks. Peaks were recorded at Dapp = 3.28 µm2 s−1 (20% of area) corresponding to dh = 138 ± 24 nm, 0.6 µm2 s−1 (75% of area) for dh = 752 ± 150 nm and 0.08 µm2 s−1 (5% of area) for dh = 5265 ± 216 nm. With the presence of NaCl, peaks were shifted towards lower values of the diffusion coefficient of 2.73 µm2 s−1 (26% area) corresponding to dh = 147 ± 20 nm, 0.26 µm2 s−1 (68% of area) for dh = 1547 ± 249 nm and 0.07 µm2 s−1 (6% of area) for dh = 5363 ± 166 nm.

Table 1 presents electrolytic conductivity and electrophoretic mobility of samples.

Table 1 Selected physical and chemical properties of HA < 30 kDa and HA > 30 kDa

The type of HA molecular fraction significantly influenced electrolytic conductivity of suspension. The conductivity was lower in HA > 30 kDa suspensions than in HA < 30 kDa. Salt caused significant increase in electrolytic conductivity. NaCl presence had significant influence in the case of both fractions for HA < 30 kDa and for HA > 30 kDa.

After 196 h, conductivity of samples was significantly lower than the one measured immediately after the preparation was made. Time had significant influence on results in the case of HA < 30 kDa fractions than HA > 30 kDa fractions.

Electrophoretic mobility of suspensions depended on the type of humic acid fraction and was higher (absolute value) for HA < 30 kDa than HA > 30 kDa. Salt, that was not significant, caused the reduction of electrophoretic mobility. Time caused significant changes of electrophoretic mobility. It was important for both fractions < 30 kDa and > 30 kDa and led to the increase in mobility.

Measurement of growth attributes

The influence of HA < 30 kDa and HA > 30 kDa on average content of fresh and dry mass of over ground parts of soybean seedlings is presented in Fig. 3a, b. Plants growing under salt stress had much smaller fresh and dry mass over ground parts comparing to the control variation. In the case of Progres, adding HA > 30 kDa significantly increased (ca 60%) fresh and dry mass of over ground parts comparing to plants under salt stress.

Figure 3c presents the influence of HA above and below 30 kDa on the height of Nawiko and Progres soybean cultivars growing under salt stress. A major drop in the height of plants was observed in both cultivars under salt stress comparing to the control group. In the case of Progres, adding HA > 30 kDa significantly increased the height of plants comparing to those under salt stress (ca 60%).

A major reduction of dry mass was observed in Nawiko under salt stress comparing to the control plants (Fig. 3d). In the case of Progres, adding HA > 30 kDa significantly increased the average dry mass of roots comparing to plants growing under salt stress.

Fig. 3
figure3

Influence of HA fractions on fresh (a) and dry (b) mass of over ground parts, height of plants (c) and dry mass of roots (d) of Nawiko and Progres soybean seedlings growing under salt stress. Average values marked with the same letters do not differ at the significance level p = 0.05; Tukey’s test; a, b—for Nawiko; A, B—for Progres

Both cultivars growing under salt stress had a lower leaf area comparing to the control plants (Fig. 4). In Progres, adding HA > 30 kDa compensated effects of salt stress and caused increase in the leaf area.

Fig. 4
figure4

Influence of HA fractions on leaf area (a) and chlorophyll content in leaves (b) of Nawiko and Progres soybean seedlings growing under salt stress. Avarange values marked with the same letters do not differ at the significance level p = 0.05; Tukey’s test; a, b, c—for Nawiko; A, B, C—for Progres

Both cultivars growing under salt stress had statistically lower content of chlorophyll comparing to control plants (Fig. 4). It was observed that in plants growing under salt stress, adding HA < 30 kDa and HA > 30 kDa caused the increase in chlorophyll content comparing to plants growing under salt stress only.

Based on biometric measurements, it was observed that salt stress reduces the length of shoots cells in both cultivars comparing to the control plants (Fig. 5). In the case of Nawiko growing in presence of NaCl, adding HA fractions above and below 30 kDa stimulated the length of shoot cells comparing to plants growing under salt stress only. In the case of Progres, adding HA < 30 kDa only increased the length of shoot cells comparing to plants growing under salt stress.

The length of stomas decreased under salt stress in Progres, whereas in Nawiko the length increased comparing to the control plants (Fig. 5). It was also determined that adding humic acids fractions below 30 kDa significantly increased the length of stomas in Progres comparing to plants growing under salt stress.

Fig. 5
figure5

Influence of HA fractions on length of shoot cells (a) and stomas (b) in Nawiko and Progres soybean seedlings growing under salt stress. Average values marked with the same letters do not differ at the significance level p = 0.05; Tukey’s test; a, b, c—for Nawiko; A, B, C—for Progres

No major changes were observed as regards the length of root cells and the number of stomas in both cultivars under salt stress and various HA fractions.

Table 2 presents the influence of salt stress with and without adding HA fractions on the content of macro-elements in over ground parts of Nawiko and Progres. In the case of Nawiko and Progres, the largest content of Mg was marked in the control variation. Salt stress caused a significant decrease in Mg content comparing to the control variation. Adding NaCl and HA fractions in parallel, only fractions above 30 kDa caused major decrease in Mg content in Progres seedlings.

Table 2 Content of macro-elements in Nawiko and Progres seedlings growing under salt stress with and without HA fractions

The Ca content in Progres growing under salt stress significantly increased comparing to the control variation. It was also observed that plants growing under salt stress after adding humic acids fractions below 30 kDa had higher Ca content. Comparing to the control variation, the Ca content in seedlings was higher by about 16%. The HA fraction above 30 kDa in combination with salt stress significantly increased the Ca content in leaves comparing to plants growing under salt stress only. In the case of Nawiko, no major influence of NaCl on Ca absorption in soybeans was found. Only in the case of combined salt stress and HA < 30 kDa the Ca content in Nawiko was lower than under salt stress only.

Nawiko and Progres growing under salt stress with and without adding HA fractions had significantly higher Na content than plants of the control variation. Only in the case of Progres, it was observed that adding HA < 30 kDa and HA > 30 kDa significantly reduced Na content comparing to salt stress only.

Table 3 Content of micro-elements in Nawiko and Progres seedlings growing under salt stress with and without adding HA fractions

The Cu content in Progres growing under salt stress was significantly lower than in the control variation (Table 3). Fraction HA, above 30 kDa, significantly reduced Cu content in leaves of the Progres cultivar comparing to salt stress only. In the case of Nawiko, no influence of salt stress was observed on the Cu content. Higher Cu content comparing to the control variation was found in leaves of plants growing under salt stress after adding HA > 30 kDa.

Under slat stress, both Nawiko and Progres showed higher Mn content comparing to the control variation, respectively about 30 and 8% (Table 3). The application of HA < 30 kDa significantly increased Mn content whereas HA > 30 kDa significantly reduced Mn content in Progres comparing to salt stress only. In Nawiko, adding HA fractions did not have a significant influence on Mn content comparing to salt stress only.

The analysis of Zn content in the over ground parts of Nawiko and Progres soybean seedlings showed that salt stress significantly increased Zn content comparing to the control variation, respectively by about 90% in the case of Nawiko and about 17% in the case of Progres (Table 3).

In the case of Nawiko, adding HA fractions above 30 kDa caused major increase in Zn content whereas in the case of Progres, major reduction of Zn content in leaves comparing to salt stress only.

The content of Fe in leaves of Nawiko significantly increased under salt stress comparing to the control variation. Adding HA < 30 kDa and HA > 30 kDa in combination with salt stress additionally significantly increased Fe level comparing to salt stress only. In the case of Progres, a reverse relationship was observed. Salt stress significantly reduced Fe content comparing to the control variation. Adding HA < 30 kDa and HA > 30 kDa also significantly reduced the Fe level in leaves comparing to salt stress only.

Discussion

Changes of physical and chemical properties of humic acids suspensions depending on salinity

Humic acids were added to the medium used in the soybean hydroponic culture to check their influence on plant growth and development in salt stress conditions. However, the salinity as well as the duration of the experiment modified also the physicochemical properties of HA.

Multimodal distributions of the diffusion coefficient, and simultaneously the size of HA particles, showed major heterogeneity (polydispersity) of suspensions (Figs. 1, 2).

The presence of particles of the hydrodynamic diameter 102 nm in both HA fractions shortly after the preparation of samples (0 h) indicates that they were already significantly aggregated. The size of humic acid particles mentioned in the literature varies from several (Thurman et al. 1982; Baigorri et al. 2007) to several dozens of nm (Baigorri et al. 2007) or several 100 nm (Palmer and von Wandruszka 2001). Baalousha et al. (2006) noted that molecules of about 140 nm are agglomerates comprising of about 20 nm units making a network. In the systems examined by us, the acidic environment of the buffer stimulated fast aggregation (Pinheiro et al. 1996). The fact, that in the diffusion coefficient distribution there were no peaks corresponding to molecules of several nanometers is not decisive regarding their absence in systems tested. For non-homogeneous samples—like these analyzed by us—large particles mask the presence of smaller ones in DLS results due to major light scattering (Filella et al. 1997).

In the case of both HA fractions, adding salt and the duration of the experiment shifted distributions of the diffusion coefficient towards lower values (Figs. 1, 2). It was the result of presence of larger molecules that showed poorer diffusion properties.

Higher electrolythic conductivity in HA < 30 kDa than HA > 30 kDa indicated the presence of smaller molecules of higher electrical charge (Table 1). The aggregation in time (or even precipitation), when observed at the distribution of the diffusion coefficient (as well as particle size), resulted in reduced conductivity.

The negative values of electrophoretic mobility resulted from dissociation of acidic functional groups (Table 1). In the case of humic acid suspensions of pH 5.59 those were carboxylic groups of pKa 3–4.4 (Muller 1996; Baalousha et al. 2006).

More negative values of mobility were recorded in the case of HA < 30 kDa than HA > 30 kDa fraction, which was e.g. the result of high density of carboxylic groups (Table 1), as confirmed by spectral analyses of samples (data not published yet). A similar relationship between electrophoretic mobility and HA molecular weight was observed by Palmer and von Wandruszka (2001).

On one hand, the increase in electrophoretic mobility between 0 and 196 h could result from a progressing dissociation of carboxylic groups, on the other hand, elimination (precipitation) from the system of some components that are sizable and have low electric charge at their surface (as confirmed by simultaneous increase in size of molecules in both fractions and slightly reduced conductivity). The aggregation of HA molecules is usually a lengthy process (Manning et al. 2000; Baalousha et al. 2006). In an acidic environment, it is however supported by a low dissociation of molecules and hydrogen bonds between them (Hosse and Wilkinson 2001; Jovanovic et al. 2013; Kloster et al. 2013).

The presence of electrolyte reduces pKa of acidic functional groups (Pinheiro et al. 1996). The addition of sodium cation to hydroponic medium lowers the HA electrophoretic mobility by neutralizing the negative charge of molecules. This promotes aggregation of molecules due to reduced electrostatic repulsion between them.

Physical and physicochemical properties of HA fractions are important for the HA interaction with the surface of plant roots. Humic acids molecules of small hydrodynamic diameter and high diffusion coefficient easily move from the bulk solution to the root zone. On the one hand, they show large negative electrophoretic mobility (high negative electrical charge) and can be repelled by the negative surface charge of roots or be bound with them due to bridging by bivalent ions present in the solution. On the other hand, considerably large molecules showing slower diffusion maintain longer contact with roots. Due to acidic functional groups, they can interact with the medium components and become a reservoir of nutrients for roots. However, in the case of precipitation, humic acids and nutrients accumulated in their aggregates may cease to be available for roots.

In both HA fractions, adding electrolyte resulted in the increase of molecules size (aggregation) and decrease in their diffusion in the solution. This, in combination with the partial neutralization of the HA negative charge and reduction of electrostatic repulsion between HA and the roots surface, can affect the plant growth in salt stress conditions.

Measurement of growth attributes

During vegetation, plants are exposed to a number of environmental stress factors that reduce their growth and yield. One of ways for improving resistance to abiotic and biotic stress can be the use of humic substances. This research examined the influence of two molecular fractions of humic acids above and below 30 kDa on growth and development of Nawiko and Progres soybean seedlings under salt stress.

The growth attributes (fresh and dry mass of over ground parts and dry mass of roots, plant height, and chlorophyll content) of soybean were significantly affected by NaCl (Fig. 3). Similar observations were documented previously for soybean (Essa 2002; Lee et al. 2010; Wei et al. 2007; Dolatabadian et al. 2011; Gawlik et al. 2014) and for cereals (Matuszak-Slamani and Brzóstowicz 2015). It was observed in this study that adding HA > 30 kDa eliminated consequences of salt stress and significantly increased fresh and dry mass of over ground parts and the height of Progres soybean in comparison to plants exposed to salt stress only (Fig. 3a–c). The stimulatory effects of humic acids have been shown in some studies to result in enhanced tolerance to salinity stress (Çimrin et al. 2010; Mora et al. 2010; Tahir et al. 2011; Aydin et al. 2012; Khalesro et al. 2015).

Promotion of root system development is the most commonly reported initial effect of humic acids on plant growth. These effects are particularly important for the adaptation of plants to adverse soil conditions, such as salinity and could be useful for the definition of rhizosphere management practices (Römheld and Neumann 2006). In the case of Progres soybean, adding HA > 30 kDa significantly increased the average dry mass of roots in comparison to plants growing under salt stress only (Fig. 3d). The enhancement of lateral or general roots increased seedling root growth and has been reported, for instance, for tomato (Canellas et al. 2011), wheat (Tahir et al. 2011), maize (Jindo et al. 2012) and pepper (Çimrin et al. 2010). Eyheraguibel et al. (2008) found that the root development induces an increase in total radicular length and enhances root surface, resulting in a better mineral nutrition.

In the condition of a saline ground, we also observed reduced chlorophyll content, which can be one of reasons of less intensive photosynthesis (Starck et al. 1995). The chlorophyll content is very sensitive to salt exposure and a reduction in chlorophyll levels due to salt stress has been reported in several plants, such as soybean (Wei et al. 2007; Lee et al. 2010) and cereals (Matuszak-Slamani and Brzóstowicz 2015). In this study the chlorophyll content significantly declined with NaCl in the case of Nawiko and Progres comparing to the control variety (Fig. 4). Adding both HA < 30 kDa and HA > 30 kDa significantly increased the chlorophyll content in both cultivars comparing to plants growing under salt stress only. The increase in the chlorophyll content under the influence of humic acids was also observed by Chen et al. (2004) in the case of soybean, melon and ryegrass; Selim et al. (2012)—potato; and Marino et al. (2010)—pear.

It is evident that there are big changes in morphology and anatomy of plants growing in saline soils. Under salt stress, the surface of Nawiko and Progres was significantly smaller comparing to the control variation (Fig. 4). In the case of Progres, adding HA > 30 kDa eliminated consequences of salt stress and caused the increase in leaf surface. The effect of salinity on leaf anatomy and area (Farshidi et al. 2012; Nassar et al. 2016) of plants had already been reported in previous works.

Adding humic acids of below and above 30 kDa stimulated the length of shoot cells in Nawiko growing in the presence of NaCl in comparison to plants growing under salt stress only (Fig. 5). In the case of Progres, only HA < 30 kDa was capable of increasing the length of shoot cells significantly comparing to plants growing under salt stress only. It was also determined that adding HA below 30 kDa significantly increased the length of stomas in Nawiko and Progres comparing to plants growing under salt stress only (Fig. 5).

The mechanism of absorbing elements by plant roots is complex and depends on e.g. cation exchange through cell membrane, intracellular transport, ions and substances exuded by roots and bacteria (Kabata-Pendias and Pendias 1999). Accessibility of the majority of micro-elements depends on pH and electrical conductivity of the soil solution (Grattan and Grieve 1999). In natural conditions, large diversity of trace elements content was observed in the dry mass, depending on species and cultivars of plants, as well as conditions of vegetation (Mehra and Farago 1994; Kabata-Pendias and Pendias 1999). The process becomes yet more complex when we consider other stress factors. Salinity of ground influences the content of particular elements in plants (Grattan and Grieve 1999; Hu and Schmidhalter 2001; Wei et al. 2007; Tunçturk et al. 2008; Matuszak et al. 2009). The relationship between salinity and content of elements in plant tissue is complex and depends on a number of factors, such as specie, development phase, vegetative organ, type and concentration of salt and environmental factors. Additionally, the effects of HS on ions uptake appear to be more or less selective and variable, and generally depends on their origin, type and concentration in the nutrient solution, pH of the medium, application and on the species and variety of plant treated (Nardi et al. 2002; Muscolo et al. 2007; Katkat et al. 2009; Celik et al. 2011; Khaled and Fawy 2011). Therefore, the literature describes a large variety of results. However, there are different ideas related to how salinity affects the micronutrient composition of plants. It was declared that the micronutrients are generally less affected by salt stress compared with macronutrients (Hu and Schmidhalter 2001). Celik et al. (2011) and Katkat et al. (2009) observed that foliar application of humic acid had a statistically significant positive effect on Cu, Zn, Mn, Mg and Fe uptake by wheat.

The research concerning the influence of NaCl and HA fractions on the content of macro and micro-elements in Nawiko and Progres leaves show differences in their accumulation (Tables 2, 3).

As expected, both cultivars of soybean growing under salt stress showed statistically significant increase in accumulation comparing to the control variation. Salt stress is known to enhance the uptake and accumulation of toxic ions such as Na+ in crop species (Essa 2002; Matuszak et al. 2009; Amirjani 2010; Doğan 2011). It was observed by us that adding both HA fractions significantly reduced sodium uptake by the Progres variety comparing to plants growing under salt stress only (Table 2).

Khaled and Fawy (2011) and Asik et al. (2009) noted that salinity decreased the uptake of nutrients except for Na and Mn. Foliar application of humic acids increased the uptake of P, K, Mg, Na, Cu and Zn. Çimrin et al. (2010) determined that the humic acid application significantly increased N, P, K, Ca, Mg, S, Mn and Cu content in the shoot of pepper seedling. They also showed that N, P, K, Ca, S, Fe, Mn, Zn and Cu contents in root increased with humic acid application. Na contents in both shoot and root of pepper decreased with increased humic acid doses. It can be concluded that high humic acid doses has positive effects on salt tolerance based on the plant growth parameters and nutrient contents.

The increased accumulation of sodium in leaves was accompanied by changes in the content of magnesium and calcium (Table 2). Decrease in the content of K, Mg and Ca due to salinity in soybean was observed by Amirjani (2010) i Essa (2002). In the case of both cultivars, humic acid fractions reduce Ca uptake in comparison to salt stress only. However, the influence of HA < 30 kDa is stronger with Nawiko and HA > 30 kDa with Progres. In the case of Nawiko, salt stress reduced magnesium uptake in comparison to the control variation (Table 2). In the case of Progres, HA > 30 kDa significantly reduced magnesium uptake comparing to the control variation and salt stress. It was observed that in the case of Progres HA reduce uptake of particular macro-elements comparing to salt stress only, chiefly HA > 30 kDa. According to Cimrin et al. (2010), Marino et al. (2010) and El-Nemr et al. (2012), the presence of humus substances increased uptake of N, P, K, Ca, and Mg.

The results related to micronutrient contents of leaves of plants are shown in Table 3. Nawiko subjected to salt stress showed significantly higher content of Mn, Zn and Fe comparing to the control variation. Additionally, the content of Zn and Fe in Nawiko leaves increased after adding HA > 30 kDa, whereas Fe increased also under the influence of HA < 30 kDa comparing the variation under salt stress only. In the case of Progres, plants growing under salt stress contained more Mn and Zn, and less Cu and Fe comparing to the control variation. Absorption of Cu, Mn, Fe and Zn significantly dropped after HA > 30 kDa was applied, whereas Fe dropped also due to the influence of HA < 30kD in comparison to plants growing under salt stress only. Solely in the case of Mn, the use of HA < 30 kDa resulted in higher accumulation.

Summarizing, we may conclude that both cultivars of soybean react strongly to salt stress. HA molecular fractions reduce or eliminate the effect of salt stress; However HA > 30 kDa is more effective than HA < 30 kDa as regards reducing negative effects. HA may stimulate shoot and root growth, and improve resistance to environmental stress in plant, but the physiological mechanism has not been well established (Delfine et al. 2005). The biochemical and molecular mechanisms underlying these events are only partially known. HS have been shown to contain auxin and an “auxin-like” activity of humic substances has been proposed, but support to this hypothesis is fragmentary (Trevisan et al. 2010).

It was also observed in the case of Progres that the uptake of macro and micro-elements in the presence of HA fractions above 30 kDa was reduced in comparison to plants growing under salt stress only. In the case of Nawiko, the uptake of macro-elements did not show significant differences. In the case of micro-elements, we observed statistically significant increase in the uptake of Fe and Zn in the presence of HA above 30 kDa.

More significant role of HA > 30 kDa than HA < 30 kDa fraction in reduction of the salt stress effects is connected with the physicochemical properties of these molecules. Particles of HA fraction with higher molecular weight were bigger and diffused slower in the solution. However, their interaction with the negatively charged surface of plant roots was easier than for HA < 30 kDa fraction due to the occurrence of lower electrostatic repulsion. The values of electrophoretic mobility of HA > 30 kDa were less negative than these of HA < 30 kDa fraction so its negative electrical charge was also lower in consequence. In the solutions of both HA fractions the aggregation process was observed during the 7 days of salt stress that could additionally affected the NaCl activity and nutrient uptake.

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Acknowledgements

This work was supported by Polish National Science Centre, project NN 310 162338.

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RM-S—designed the research, conducted experiments and analyzed data, wrote the manuscript; RB: HA extraction, HA separation, analyzed data, wrote the manuscript; JC—conducted experiments and analyzed data, wrote the manuscript; AB, DG—designed the research; MK, AG, DK, MW—conducted experiments; MS: HA extraction, HA separation. All authors read and approved the final version of the manuscript.

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Correspondence to Renata Matuszak-Slamani.

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Matuszak-Slamani, R., Bejger, R., Cieśla, J. et al. Influence of humic acid molecular fractions on growth and development of soybean seedlings under salt stress. Plant Growth Regul 83, 465–477 (2017). https://doi.org/10.1007/s10725-017-0312-1

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Keywords

  • Humic acids
  • Hydroponics
  • Nutrient uptake
  • Plant growth
  • Salinity
  • Soybean [Glycine max (L.) Merr.]