Physiological markers of stress susceptibility in maize and triticale under different soil compactions and/or soil water contents

ABSTRACT Differences between two maize and two triticale genotypes grown in low soil compaction (LSC), moderate soil compaction (MSC) and severe soil compaction (SSC) and with a limited (D) or excess (W) soil water content were observed as a decrease in shoot (S) and root (R) biomass, leaf greening (SPAD) and increase in membrane injury (LI), root and leaf water potential (ψ), photosynthesis (Pn), transpiration (E) and stomata conductance (gS). Close correlations between ψL and ψR, and between differences ψL and ψR (Δψ) were found. Drought or waterlogging with LSC conditions in both maize genotypes resulted in higher WUE than in control plants (LSC C), but under the SSC WUE declined. However, for triticale differences in WUE, between treatments were small and insignificant. In general, changes in markers were greater for genotypes sensitive to the soil compaction (Ankora, CHD-12) than in resistant ones (Tina, CHD-247) and were higher in seedlings grown under SSC conditions. Abbreviations: ψR, ψL: root and leaf water potential; C: control; D: drought; E: transpiration rate; FWC: field water capacity; gS: stomatal conductance; LSC, MSC, SSC: low, moderate and severe soil compaction; Pn: photosynthesis rate; W: waterlogging


Introduction
The conservation of water in agriculture requires an understanding of the mechanisms of soil-plant-water relations. Drought, waterlogging and soil degradation are environmental stresses caused by the climate changes (Passioura et al. 1993;McKersie and Leshem 1994, Kozlowski 1999, Masle 2002, Ehlers and Goss 2003, Ashraf 2010. Cell dehydration caused by a soil water deficiency or excess impairs various physiological processes, especially, changes in a plant growth, development and productivity. It is known that during soil water stresses C4 plants are often more competitive than C3 plants (Colombi and Walter 2016). The responses to the decreased leaf water potential are functional and structural changes in chloroplasts, limited CO 2 diffusion to chloroplasts and disturbances in accumulation and distribution of assimilation products (Cornic and Massacci 1996;Flexas and Medrano 2002;Medrano et al. 2002;Nayyar and Gupta 2006;Ripley et al. 2007;Ghannoum 2009). Physiological processes are affected also by a combination of different abiotic environmental stresses, including compacted soil and soil water deficiency or excess. Excessive soil compaction results from natural processes, past glacial ice pressure, by soil settling, slumping and the use of heavy equipment in soil cultivation. Increase in soil compaction causes changes in periodic soil mechanical impedance, hydraulic permeability, air conductivity and diffusivity and slower rates of water infiltration, resulting in risks of limited or excess soil water content (Boyer 1982;Iijima et al. 1991;Kozlowski 1999, Masle 2002Ashraf 2010;Chan and Weil 2010;Grzesiak et al. 2014). The main effect of high compaction are changes in a root structure (number, length, thickness, direction of growth in soil) and in the above-ground plant parts (plant height, stem diameter, leaf number, leaf thickness and area, specific leaf area, thickness of epidermal cell and cell wall) (Yamauchi 1993;Clark et al. 2003;Fageria et al. 2006;Mommer et al. 2006). In the case of drought, the amount of rainfall does not compensate water loss through transpiration and evaporation, and in the case of waterlogging, the soil is inundated as a result of heavy rainfall or river floods, which cause a drastic decrease in roots' capability for water uptake as a result of decreased oxygen content in water. Excess of water in the soil destroyed the water balance in plants, resulting in oxygen limitation in plant roots (Haupt-Herting and Fock 2002;Mommer et al. 2006;Frost-Christensen and Floto 2007). With the increasing water stress, the absence of oxygen made the plant roots rotting ultimately, and the above-ground parts wilting, which even resulted in death of plants (Lawlor and Tezara 2009;Chan and Weil 2010). Negative water balance in plant tissues is one of the common consequences of environmental stresses to which plants are exposed and as such is the bottleneck of agricultural progress.
The first responses of plants to those stresses are changes of tissues' water content, membrane permeability, chlorophyll content and gas exchange parameters (Palta 1990;Ripley et al. 2007). Exposure of roots to oxygen shortage by waterlogging induces changes in the dark respiration, use of carbohydrates, the synthesis of antioxidants and the induction of glycolysis and fermentation. Plants adapt to hypoxia by metabolic processes such as maintaining carbohydrate content, avoiding acidification of the cytoplasm and launching a defence antioxidant system. It has also been shown that plants transition to anaerobic respiration in order to meet the demand for energy under oxygen-deficiency conditions as a result of blocking the Krebs cycle and oxidative phosphorylation (Lipiec et al. 1996;Crawford 2003;Gibbs and Greenway 2003;Tubeileh et al. 2003;Sairam et al. 2008;Rut et al. 2010;Sun et al. 2015).
Despite the amount of information about processes happening during growth under soil compaction is relatively little (Tracy et al. 2015), drought and waterlogging have been studied as separate stress factors in different plant species and cultivars. The tolerance of plants to different stresses is determined by the plants genes and the degree of plant restriction and depends on the species, variety and age of the plants. Stress susceptibility indexes are used for description and explanation of strategies implemented by plants to alleviate and/or remove an environmental stress influence (Blum 1996;Golbashy et al. 2010;Liu et al. 2010;Grzesiak et al. 2012Grzesiak et al. , 2013. The effect of stomatal or non-stomatal factors on photosynthesis and transpiration was analyzed in many studies; however, the conclusions from these studies are contradictory. Both stomatal and non-stomatal mechanism could be involved in the changes of leaf gas exchange parameters during soil water stress. Some researchers observed that stomatal behavior was the major factor limiting photosynthesis but on the other hand, other authors showed that a decrease in photosynthetic rate was due to non-stomatal factors (Berkowitz et al. 1983;Cornic and Briantai 1991;Bethenod et al. 1996;Cornic and Massacci 1996;Dubey 1997;Cornic and Fresnau 2002;Tubeileh et al. 2003;Kebbas et al. 2015). Index of water use efficiency (WUE) is defined as the ratio of dry mass accumulation to plant water use or by ratio of photosynthesis rate to transpiration rate. Improvements in the WUE of rainfall and irrigation could play a key role in agricultural practices because it often correlates with environmental stress tolerance. Also, an important factor influencing WUE is stomata because stomata play a very crucial role in the uptake of CO 2 and H 2 O vapor exchange (Kriedemann and Dowton 1981;Baker 1993;Passioura et al. 1993;Ashraf and Arfan 2005;Fageria et al. 2006). During water stress, application of the soil-root-shoot water balance is essential for plant survival and growth. A number of studies have reported that soil water stress stimulates the production of hydraulic and chemical signals in the roots of plants, which are then transported to the leaves to regulate stomatal aperture. According to , soil compaction affects seedlings' root anatomy (xylem vessel diameter and proportion) and morphology (specific root length), and this influences gas exchange parameters (photosynthesis and transpiration rates). Disturbances of the photosynthesis at the molecular level are connected with the low electron transport through PS II and/or with structural injuries of PS II and LHC complexes (Tardieu et al. 1991;Smith and Griffiths 1993;Jackson 2002;Jackson and Ram 2003;Asch et al. 2009;Else et al. 2009).
The aim of this study was to examine the response of maize and triticale genotypes (differing of susceptibility to soil compaction), grown in different soil compaction levels and with limited or excess soil water content. Maize and triticale are of the most important cereals in the world and is cultivated under a wide range of climatic conditions. Both species are an interesting research model because each has, for example, a different photosynthesis pathway, bundle sheath structure (Kranz-type syndrome) and root system structure (scattered, concentrated). The degree of susceptibility of maize and triticale to the combined stresses of soil compaction with drought or waterlogging is a desirable physiological research target, and discovering the physiological mechanism and exploring strategies conferring cross-tolerance are important agronomic interests. Determination of physiological markers (membrane injury, leaf greening, gas exchange, water potential of root and leaf) may explain how cereals manage its growth under multistress environment.

Plant material
Two maize single-cross hybrids (Ankora, Tina) obtained from SEMPOL-Holding Trnava, Slovakia and two and two triticale breeding form (CHD-12, CHD-247) obtained from breeding station Małyszyn (Poland) were used in the experiment. These maize and triticale genotypes had been used in our previous studies (Grzesiak et al. 2014), on the basis of which Ankora and CHD-12 were selected as susceptible to soil compaction stress and Tina and CHD-247 as resistant (Table 1).

Growth conditions and experimental treatments
Plants were grown in an air-conditioned greenhouse under the following day/night conditions: temperature 23°C/18°C (±2.5°C) and relative humidity 70%/60% (±5%), during a 14-h photoperiod from 7 am to 9 pm (artificial irradiance from high pressure sodium lamps, Philips SON-T AGRO, 400 W). Photosynthetically active radiation (PAR) was equal to about 350 µmol m −2 s −1 .
One pre-germinated grain was planted per pot at a depth of 2 cm. Pots used in the experiments were PVC tubes of 11 cm diameter and 18 cm height, fitted with a window which enabled the sampling of roots. Pods were filled of quartz sand (fraction < 1mm) as rooting medium produced by AQUAEL Ltd. (Poland). Air-dried sand was sieved with 0.25 cm mesh (to remove fraction larger than 2.5 mm) and mixed with compound fertilizer: N -28 mg, P -18 mg, K -14 mg per 1 kg. Three soil substrate compaction treatments were appliedlow (LSC -1.1 g cm −3 ), medium (MSC -1.3 g cm −3 ) and severe (SSC -1.6 g cm −3 ). Sand was compacted in pods by using hydraulic press. Soil substrate mechanical resistance was measured with the penetrometer DIK 5520 (Daiki Rika Kogyo Co. Ltd., Japan).
Field water capacity (FWC) of soil substrate was determinate according to Hillel and van Bavel (1976). Air-dried samples of 100 cm 3 compacted to the three impedance values weighed 110.0, 130.0 and 160.0 g. These were placed inside metal cylinders, with a 1-mm hole at the bottom. The cylinders of sand were placed inside a container with water for 30 min. After 8 h, maximal soil water content in samples was 0.47, 0.41 and 0.39 g cm −3 , and after 48 h it decreased to 0.25, 0.22 and 0.18 g cm −3 , respectively. According to the latter, values were assumed to be 100% of FWC. During the experiments the PVC tubes were weighed every day, and the amount of water loss through evapotranspiration was refilled to keep the constant mass in each treatment. For control treatments (LSC C, MSC C and SSC C), soil water content was maintained at 65-70% FWC from sowing to 28th day. In drought treatments soil water content was kept at 30-35% FWC, and the pots were not watered for 14 days from 14th till 28th day (LSC D, MSC D, SSC D). Similarly, for waterlogging, soil water content was kept at 100% FWC from 14th to 28th day (LSC W, MSC W, SSC W). For waterlogging PVC tubes were submerged in a container in which the water surface was 1 cm above the soil surface.

Biomass
Dry matter of the above-ground parts (S) and roots (R) was determined after drying at 65°C for 72 h.
Membrane injury index (LI) was determined as relative loss of intracellular electrolytes from leaf tissues and was measured with the conductivity method using conductivity meter OK-102/1 (Radelkis, Hungary), according to the procedure described by Blum and Ebercon (1981).
where C and T refer to the conductivity of control and treatment solutions, respectively, and subscripts 1 and 2 refer to initial and final conductivity, respectively. Nine leaf discs (0.5 cm diameter) were cut from leaves and immersed in test tubes containing 30 cm 3 redistilled water. After 24 h, initial conductivity measurements were taken. Final conductivity measurements were taken after autoclaving all tubes at 110°C for 15 min and cooling them to the room temperature. Water potential (ψ) was measured with psychrometer HR33T (Wescor Inc., Logan, USA) in 'dew point' mode, equipped with sample chamber C-52 SF (Wescor Inc., Logan, USA) and digital multimeter Metex M-3640 D. Samples were placed inside the psychrometer chamber and left to balance temperature and water vapor equilibrium for 30 min before measurements. Measurements were made on the fourth, i.e. most recent fully expanded leaf.
Leaf greening in SPAD units was measured with CL 01 meter (Hansatech, Norfolk, UK).

Gas exchange
Rate of net photosynthesis (P N ), transpiration rate (E) and stomata conductivity (g s ) was measured using IRGA analyser (CIRAS-2, PP System, Amesbury, USA) with a Parkinson's assimilation chamber (narrow leaf type) and with light attachment. During the measurements, an open system was used. The flow rate of ambient air with a constant CO 2 concentration (390 µmol mol −1 ) through the assimilation chamber amounted to 0.5 dm 3 min −1 . Chamber temperature was kept below 25°C until the photosynthesis rate stabilized.
Photosynthetic capacity at light saturation was reached by exposing leaves to PAR at 800 µmol m −2 s −1 . Index of WUE was calculated as the ratio of Pn to E. Measurements were made on the fourth leaf from 11 am to 1 pm in nine replications.

Statistical analysis
The experiments were performed in a completely randomized design. The results presented are mean values on nine (Dry matter, LI, SPAD, Pn, E, g S ) and six (ψ R and ψ L ) replications. Data were analyzed with the statistical package STATISTICA 12.0 (Stat-Soft Inc., Tulsa, OK, USA) using analysis of variance (ANOVA) and Duncan's multiple range test at p ≤ .05.

Mechanical impedance of soil substrate
For Both an air-dried and a wet soil in all soil compaction treatments (low-LSC, moderate-MSC and severe-SSC), soil mechanical impedance was increasing with soil depth, and its mean value for LSC, MSC and SSC of air-dried soil was 0.85, 1.23 and 2.01 MPa, respectively, and for wet soil (65% FWC), it was 0.74, 1.05 and 1.67 MPa, respectively. Differences between air-dried and wet soil in MSI and treatments LSC, MSC and SSC were 0.09, 0.18 and 0.34 MPa, respectively.

Effect of separate application of soil compaction stress on measured traits
Both maize and triticale seedlings' growth under moderate (MSC) or severe (SSC) soil compaction, in comparison to low soil compaction (LSC), resulted in a decreased dry matter of S and R and changes in S/R ratio (Table 2). After 28 day of growth in MSC and SSC treatments, the dry matter of S decreased in maize hybrids Ankora to about 4% and 16% and in hybrids Tina to 3% and 14%, respectively. Similarly, in triticale, genotype CHD-12 decrease in S was found to be about 9% and 17%, respectively, and in genotype CHD-247 to 7% and 10%, respectively. Dry matter of roots (R) decreased in the MSC and SSC treatments in Ankora about 7% and 26%, respectively, while for the Tina were 4% and 16%. For CHD-12, the decrease was 14% and 29% and for CHD-247 to 7% and 16%, respectively. Significant increase in the S/R ratio to about 13% and 17% was observed only for Ankora and CHD-12 grown under SSC. Differences between resistant and sensitive genotypes were observed in membrane injury (LI) and chlorophyll content in SPAD units (Table 3). Growth under increased soil compaction Table 1. Soil compaction stress susceptibility (SSC SI), soil root penetration ability (RPA) and the root penetration ability through a petroleum-wax layer indexes in maize and triticale genotypes according to Grzesiak et al. (2014 caused a raise in LI and decrease in SPAD, and observed changes were higher in sensitive genotypes. Correlation coefficients (r) between total seedlings dry matter (S + R) and membrane injury (LI) and leaf greening (SPAD) were high and statistically significant (−0.769 and 0.907 for maize and for triticale −0.847 and 0.932, respectively).
Measurements of leaf (ψ L ) and root (ψ R ) water potential and gas exchange parameters (Pn, E, g S , WUE) were made only for low (LSC) and severe (SSC) soil compaction levels (Tables 4-8). In control seedlings grown under SSC treatment, decrease in ψ R and ψ L between 3 and 14 days were slightly larger than in seedlings grown under LSC treatments. Mean decrease of ψ R and ψ L , in maize genotypes Ankora were about 148% and 130%, respectively, and in Tina about 132% and 124%, respectively. For triticale: CHD-12 was about 153% and 137% and in CHD-247 about 132% and 128%, respectively (Table 4). Similarly, a decrease in gas exchange parameters in genotypes grown under SSC treatments in comparison to LSC treatments was observed (Tables 5-8). Mean decrease of Pn was in Ankora about 17% and Tina 12%, CHD-12 about 13% and CHD-247 about 10%. The decrease of transpiration rate (E) in Ankora and Tina was about 12% and 6%, respectively, and for CHD-12 and CHD-247 was about 10% for both genotypes. Similarly, a decrease of mean stomata conductivity (g S ) was observed. Smaller differences, often not statistically significant between plants grown in the conditions of the LSC and SSC, were observed in WUE calculated as a ratio of Pn to E. In general, differences between genotypes susceptible to soil compaction were only slightly greater than for resistant ones but in many cases were not statistically significant.

Changes of biomass
The soil compaction stress (SC) combined with soil drought (D) or waterlogging (W) influenced a dry matter of shoot (S), roots (R) and shoot to roots (S/R) ratio ( Table 2). The obtained results show that decrease of S and R in seedlings grown under MSC and SSC soil compaction in comparison with LSC were grater in seedlings grown under drought (D) than under waterlogging (W) and were higher in seedlings grown under SSC for sensitive genotypes (Ankora, CHD-12) than resistant genotypes (Tina. CHD-247). Increase of S/R ratio was observed only in both maize hybrids (Ankora, Tina) and the sensitive triticale genotype (CHD-12). The ANOVA test shows significant differences for treatments (T) genotypes (G) for S, R and S/R ratio. Also significant differences were observed for interaction T × G, for S, R and S/R except triticale.

Membrane injury (LI) and chlorophyll content (SPAD)
Measurements of LI and SPAD were carried out in seedlings grown in three levels of soil compaction (LSC, MSC, SSC) and subjected to drought (D) or waterlogging (W) stresses from Table 2. Changes in dry weight of shoot (S), root (R) and ratio of dry weight of shoot to root (S/R) seedlings of maize and triticale genotypes grown 28 days under three soil compaction level (low -LSC, moderate -MSC and severe -SSC) and 14 days under, drought (D) or waterlogging (W), and ANOVA for measured traits.
Note: Mean value n = 9; nsnot significant. *Statistically significant at a level of probability p < .1. **Statistically significant at a level of probability p < .05. ***Statistically significant at a level of probability p < .01.
14th to 28th day of growth (Table 3). Membrane injury (LI) of maize and triticale seedlings grown under increased soil compaction levels and drought (D) were higher than in waterlogging (W) conditions. Differences between sensitive and resistant genotypes were observed in seedlings affected by drought or waterlogging. Membrane injury (LI) in treatments MSC D and SSC D in comparison with LSC D increased respectively in Ankora about 40% and 120%, in Tina about 15% and 85%, in CHD-12 about 40 and 135% and in CHD-247 about 40 and 160%. The ANOVA show statistically significant variance for all factors (T, G) and interactions between T × G (Table 3).

Chlorophyll content (SPAD)
For seedlings grown under three soil compaction levels and subjected to drought (LSC D, MSC D, SSC D) or waterlogging (LSC W, MSC W, SSC W), a decrease of chlorophyll content in SPAD units was observed (Table 3). Under control soil water content the decrease in SPAD in MSC C and SSC C treatments in comparison with LSC C was in Ankora about 25% and 58%, respectively, in Tina about 25% and 42%, in CHD-12 about 15% and 35% and in CHD-247 about 14% and 27%. Decrease in SPAD for seedlings grown under drought (D) was greater in comparison with seedlings grown under waterlogging (W). The significant differences between sensitive and resistant genotypes of maize and triticale were observed in all treatments and all soil compactions except Ankora and Tina grown under low soil compaction. Table 3 shows the ANOVA of SPAD. For all factors (T, G) and all interactions were (T × G) found statistically significant variance.
Root (ψ R ) and leaf (ψ L ) water potential Measurements of ψ R and ψ L were carried out after 3, 7, 10 and 14 days of growth under low (LSC) and severe (SSC) levels of soil compaction for seedlings grown under control conditions (LSC C, SSC C) and for drought (LSC D, SSC D) or for seedlings flooded (LSC W, SSC W). Samples of leaf and root were taken from 11 am to 13 pm in three replications. The reason for the changes of ψ R and ψ L under drought and around noon hours is that the high rate of transpiration at midday is not counterbalanced completely by the roots' water uptake from the soil (Table 4). Under control treatments, differences between sensitive and resistant genotypes in ψ R and ψ L were small and insignificant; however, significant differences were observed between seedlings grown under low and severe soil compaction (SSC). Both in maize and triticale genotypes grown in SSC, decrease of ψ R and ψ L were greater than in seedlings grown in low soil compaction. For seedlings subjected to drought (D) or waterlogging (W), decrease of ψ R and ψ L in comparison with controls treatments was observed. Table 3. The leaf membrane injury (LI)* and leaf greenness (SPAD) in maize and triticale genotypes grown from sowing to 28 day in low (LSC), moderate (MSC) and severe (SSC) levels of soil compaction and with 14 days (from 14 to 28 days) of soil drought (D) or waterlogging (W) and ANOVA test of significance for measured traits. Source of variance LI SPAD LI SPAD Treatment (T) 5 *** *** *** *** Genotype (G) 1 ** ** ** ** T × G 5 * ** *** ** Notes: Mean values (n = 9) ± standard error. Treatment LSC C was used as control for treatments MSC C and SSC C, respectively, and treatments LSC C, MSC C and SSC C were used as controls for treatments with drought (D) or waterlogging (W), respectively. *Statistically significant at a level of probability p < .1. **Statistically significant at a level of probability p < .05. ***Statistically significant at a level of probability p < .01. Table 4. Changes of leaf (ψ L ), root (ψ R ) and Δψ water potential in successive days of applied drought (D) or waterlogging (W) in two maize genotypes grown under low (LSC) or severe (SSC) soil compaction levels and the ANOVA test.

Treatment Genotype
Leaf water potential (ψ L ) Root water potential (ψ R ) Day   mean   Day   mean  3  7  10  14  3  7  10  14  3  7 10 14   In sensitive genotypes (Ankora, CHD-12), changes were greater than for resistant genotypes (Tina, CHD-247). During successive days when drought or waterlogging stresses were applied and in both soil compaction levels differences between ψ L and ψ R were observed but under waterlogging conditions those differences were lower in comparison with drought conditions. Table 4 shows the ANOVA of root (ψ R ), (ψ L ) and (Δψ). For all factors (T, G, D) and interactions (T × G, T × D, G × D) statistically significant variance were found. However, interaction between (T × G × D) was significant only for (ψ R ) in maize.
Gas exchange parameters (Pn, E, g S ) and WUE Schedule of measurements of gas exchange parameters was same as measurements of water potential. Differences between sensitive and resistant of maize and triticale genotypes were observed in Pn, E and g S (Tables 5 and 7). Changes of gas exchange parameters were greater in seedlings grown under drought connected with SSC in comparison with seedlings subjected to waterlogging. In maize, decrease in Pn between 3 and 14 days in seedlings subjected to drought (D) was in treatment LSC and SSC about 20% and 57% for Ankora and about 16% and 43% for Tina, respectively. Similarly, in seedlings subjected to waterlogging (W), decrease of Pn in treatments LSC and SSC was for Ankora about 13% and 41% and for Tina 5% and 21%, respectively. In triticale, changes of Pn in this period were similar and in treatments LSC and SSC with drought (D) were in CHD-12 about 27% and 58% and in CHD-247 about 21% and 34%, respectively, and with waterlogging (W) in CHD-12 about 13% and 29% and in CHD-247 about 8% and 17%, respectively. As in the case of photosynthesis rate (Pn), similarly effects of soil compaction and soil water content on transpiration rate (E) and stomatal conductance (gs) were observed. Decrease of E between 3 and 14 days was greater in seedlings subjected to drought (D) than for seedling subjected to waterlogging (W) especially in seedling growing in SSC. In sensitive genotypes (Ankora, CHD-12), differences of E between control seedlings (LSC C, SSC C) and seedling subjected to drought (LSC D, SSC D) or waterlogging (LSC W, SSC W) were larger than in resistant genotypes (Tina, CHD-247). Similar changes were also observed in stomata conductivity (g S ). In seedlings growing under drought (D) stress decrease, g s was greater than in seedlings grown under waterlogging (W) and was visible especially in sensitive genotypes. Somewhat differently proceeded changes of WUE in maize and triticale. Under conditions of low soil compaction in both genotypes of maize from 3 to 10 days, WUE was higher than in control plants but after 14 days decline of WUE was observed. However, under the SSC, WUE decline compared to control was observed after 7 days and it was particularly large in the seedling grown under drought. In both genotypes of triticale grown under low soil compaction, differences between treatments were statistically insignificant. In conditions of SSC in sensitive genotype (CHD-12), WUE decreases from 7 days especially for seedlings treated with drought, but in resistant genotype (CHD-247), differences between treatments were smaller. The results of ANOVA of gas exchange parameters and WUE are presented in Tables 5-8. For maize, the variance for Pn, E, g s and WUE was significant for all variables (T, G, D) and for the all interactions. Similarly, for triticale, significant variances were found, except for stomata conductance (T × D, T × G × D) and WUE (T × G × D).
Note: Mean values (n = 9). *Statistically significant at a level of probability p < 0.1. **Statistically significant at a level of probability p < 0.05. ***Statistically significant at a level of probability p < 0.01.
Mean values (n = 9). *Statistically significant at a level of probability p < 0.1. **Statistically significant at a level of probability p < 0.05. ***Statistically significant at a level of probability p < 0.01.
Note: Mean values (n = 9); ns: not significant. *Statistically significant at a level of probability p < 0.1. **Statistically significant at a level of probability p < 0.05. ***Statistically significant at a level of probability p < 0.01.

Correlation and regression between the measured physiological markers
In this experiment, between two species, four genotypes and two levels of soil compaction conditions differences of the correlation between the studied traits were found (Table 9). In both genotypes of triticale, statistical significant correlations were observed in plants grown under SSC in all cases, except for WUE with E. On the other hand, in the seedlings grown under low soil compaction (LSC), significant coefficients were not observed at a resistant genotype (CHD-247) between WUE with Pn, gs, ψ L , ψ R and ψ L − ψ R and in sensitive genotype (CHD-12) only for WUE with E and gs. In contrast, in both maize genotypes, there has been less statistically significant correlation coeficients than in the case of triticale. This mainly concerns the associations between WUE and gs with all the cases. In experiments we found not only a close association, between ψ L , and ψ R , but also differences between ψ L and ψ R (Δψ).
Soil compaction stress had a significant effect on changes in the stem and root biomass and all measured physiological traits except stomata conductance (g S ), leaf and root water potential (ψ L , ψ R ). Results presented in Figure 1 show relations between changes in total seedlings dry weight (S + R) and soil compaction, drought and waterlogging stresses. Regression coefficient (R 2 ) for relation between seedlings  Relationship between seedlings dry matter (S + R) and gas exchange parameters (Pn, E, g S , WUE) and leaf (ψ L ), root (ψ R ) water potential and Δψ (ψ R −ψ L ) of maize and triticale genotypes grown under two levels of soil compaction (LSC, SSC) and after 14 days of drought (D) or waterlogging (W). Regression coefficients (R) are shown with statistical signification: ns: non-significant; *,**,***statistically significant at p < .1, .05 and .01, respectively. dry matter and membrane injury (LI) was high, but statistically insignificant, both for maize (Figure 1(a-c)) and triticale (Figure 1(c-e)). However, for the leaf greening (SPAD), regression coefficients were statistically significant for drought and waterlogging stresses (Figure 1(h-l)) and for soil compaction only for triticale (Figure 1(j)). In Figure 2, results of linear regression between changes of total seedlings dry weight (S + R) and measured physiological traits (gas exchange parameters, water potential) are presented separately for soil compaction, drought and waterlogging stresses. Regression coefficients (R 2 ) between changes of seedlings dry matter and measured physiological traits were statistically significant only for Pn, WUE and Δψ (Figure 2(a-c,j-l,t-w)).

Discussion
Our results (Grzesiak et al. 2013(Grzesiak et al. , 2014 and results of others authors (Jones et al. 1989;Blum 1996;Lipiec et al. 1996;Clark et al. 2003;Mommer et al. 2006;Asch et al. 2009;Sun et al. 2015) indicated that the soil compaction, drought and waterlogging stresses had caused significant changes on biomass of roots (R), shoots (S), shoot to root ratio (S/R), membrane injury (LI), chlorophyll content (SPAD), leaf (ψ L ) and root (ψ R ) water potential, gas exchange parameters (Pn, E, g S ) and water use efficiency (WUE). Those stress factors are multidimensional environmental events that have considerable property on plant growth, development and yield. The response of cereals species to the combined effect of different soil compaction with soil drought or waterlogging depends on a level of soil compaction and a soil water content. Maize and triticale genotypes (Tina, CHD-247) used in this experiment, which were resistant to soil compaction, were found to be resistant also to drought or waterlogging but there are cereal species (rice) which are resistant to drought but sensitive to waterlogging (Kono et al. 1987;Iijima et al. 1991;Yamauchi 1993). Our results suggest that the reduction in plant biomass grown under different soil compaction levels is related to the degree of susceptibility to stresses (Tables 1  and 2). Similarly, we found interaction between soil compaction and drought or waterlogging for membrane injury and leaf greening. The ability to maintain the membranes' structure and function under water deficit is an important physiological marker and was used as a screening test for the estimation of tolerance to various stresses (Blum and Ebercon 1981;Palta 1990). Our results (Table 2) indicate that differences between sensitive and resistant genotypes might originate from the fact that stress-resistant genotypes possess efficient mechanisms protecting membrane functions and structure. Similarly other authors suggest that resistant plant species show stronger binding of chlorophyll molecules to the lipid-protein complex of chloroplast membranes (Smirnoff and Colombe 1988;Yu et al. 2008). In the present work, it was shown that under both different soil compaction levels and their multistress influence, together with drought or waterlogging, the decrease in SPAD was greater for the sensitive genotype. The obtained results are in compliance with the findings with studies by other authors, which showed a decrease in chlorophyll content in leaves under stress conditions (Damanik et al. 2010;Zhao et al. 2014). However, the results do not provide clear answers, confirming the relationship between chlorophyll content and the rate of photosynthesis. It was shown that a negative or positive correlation between chlorophyll content and the rate of photosynthesis is likely caused by the fact that not all of the chlorophyll present in the plant participates in photosynthesis and it may also perform other protective functions, with higher content often observed in plants exposed to stress. The reduction in chlorophyll content is caused by the inhibition of synthesis and accelerated decomposition of chlorophyll, and stressresistant species have stronger chlorophyll molecules' association with lipid-protein complex membranes of chloroplasts (Poljakoff-Mayber 1981;Smirnoff and Colombe 1988;Palta 1990). In the present paper, we show that the difference between water potential of leaf and root in seedlings subjected to different soil moisture levels lies in the limitation of water uptake by roots under drought or under waterlogging (Table  4). In the resistant genotype to compacted soil (Tina, CHD-247), the differences between ψ L and ψ R were smaller than in the sensitive one (Ankora, CHD-12). According to Tardieu (1993) and Ehlers and Goss (2003), in plants grown without watering under given evaporative demand of the air, water potential within the soil-plant-atmosphere continuum is dependent on the soil water potential. Extraction of water from defined soil volume in an experimental pot causes the soil matrix potential to decrease steadily from day to day. When ψ R and ψ L decline at noon, the tissues turgor is reduced. This will in turn affect the rate of cell enlargement, which may decline noticeably in periods of restricted water budget. Only in the night after relaxation the rates of cell enlargement and growth will increase again. Growth by cell elongation can be continued throughout the night and possibly the growth at night is greater than during the day. During the course of water extraction and soil drying in the pot, the daily amplitudes of ψ L and ψ R become more pronounced. In seedlings subjected to prolonged stresses, the critical value of water potential is reached and leaf wilting occurs because ψ L does not rise above the wilting point (Passioura et al. 1993;Smith and Griffiths 1993;Lipiec et al. 1996;Bengough et al. 2011). Also, daily changes in leaf water status of seedlings grown under high soil compaction indicate damage to light-harvesting mechanisms in stressed plants and lend support to the hypothesis that early damage to PSII explains the prompt closing of stomata by stressed plants (Jackson and Ram 2003).
The relation between leaf water content and gas exchange parameters was studied in many papers as the basis for the estimation of photosynthesis limitation by stomatal or nonstomatal mechanisms in plants grown under stress conditions. The physiological mechanisms involved in stomata behavior under different soil compaction and soil water deficiency or excess are not well explained, but some studies have shown that stomata aperture controls leaf water deficit and may improve leaf WUE. Stomata play an important role in controlling CO 2 and H 2 O vapor exchange and stomata aperture effects of Pn and E (Nilson and Assmann 2007, Lawson and Blatt 2014. The earliest response to leaf water deficit is stomata closure, which limits CO 2 diffusion to chloroplasts. Non-stomatal mechanisms under prolonged water deficit in leaf tissues include changes in chlorophyll synthesis, functional and structural changes in chloroplasts and also disturbances in accumulation and distribution of assimilation products (Kicheva et al. 1994;Medrano et al. 2002). It has been found that both electron transfer and CO 2 fixation are affected under water stress (Chaves et al. 2002;Liu et al. 2010). Moreover, during leaf water deficit, disturbances of photosynthesis at the molecular level are connected with low electron transport through PSII and/or with structural injuries of PSII and LHC complexes. As such, the decrease in Pn can be attributed to the influence of soil compaction on soil aeration and reduction of air transmission in the root system. Similar to our results, significant correlation coefficient was also found between stomata conductance and leaf water potential for flooded tomato plants (Tardieu et al. 1991;Tardieu 1993;Sobeih et al. 2004;Else et al. 2009).
In both maize genotypes, drought or waterlogging with LSC condition caused WUE to be higher but under the SSC, was lower than the control plants (LSC C). However, for triticale, differences in WUE between treatments were small and insignificant. Breeding of stress-resistant cereal species which apply water-saving strategies used to improve their WUE. Also, as a result of various stress factors, rapid changes occur in hormone levels of plant tissues, which alter the balance between synthesis, degradation and transport of hormones. Some of these changes may be adaptive responses to stressful conditions while others may be an expression of metabolic disorders (Chaves et al. 2002;Fageria et al. 2006;Golbashy et al. 2010;Liu et al. 2010;Sun et al. 2015).
Most research on soil compaction considers this stress in isolation; however, far less studies investigated interactions with other environmental stresses. Results obtained in our experiments show that effects of soil compaction on seedlings growth and physiological traits depend on the plant species and soil water availability. Soil compaction, soil drought and waterlogging were considered to have an effect on physiological processes; however, it is complicated to explain the impact of soil compaction (mitigation or aggravation of the harmful effects) with the presence of soil water stress factors. Our results show that changes in the dry matter of shoot and roots under different soil compaction strongly depend on soil water content and on interaction with a drought or waterlogging. Soil compaction stress decreases plant biomass, but it is not consistent with other research, which indicated that conditions of intermediate compaction stimulated a biomass growth because under these conditions there is better soilroot contact, which improves water and nutrient uptake (Masle 2002. In natural environments, we frequently have to deal with the situation of simultaneous presence of different stresses and their interactions cannot be directly extrapolated from the response of plants to a single stress. The combination of two or more stresses should be regarded as a new state of influences on plants, which requires a new defence or acclimation response Mittler (2006). Physiological markers assist the selection of plants with particular characteristics, but this task is time-consuming, requiring much experience, taking into account the different phases of growth and development of plants and reproducible environmental conditions. Our research and studies by other authors have shown that physiological markers are satisfactory for the study of populations in terms of their sensitivity to stress and they may support molecular testing (Masle 2002;Mittler 2006).

Conclusions
Tolerance to a combination of different stress conditions, particularly those that mimic field environment, should be the focus of research programs aimed at developing new crop genotypes with enhanced tolerance. The impact of combined stresses on the physiology of crop plants is key to understanding stress susceptibility mechanisms under natural field conditions. Recent studies have revealed that the response of plants to a combination of two different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each stress applied individually. Exposure of plants to more abiotic stresses causes most often a more harmful effect comparing to a single stress. In our study we found that responses of maize and triticale genotypes to soil compaction stress with drought or waterlogging are associated with plant water status, which is manifested in the changes of physiological traits such as membrane permeability, chlorophyll content and leaf gas exchange. Differences between sensitive and resistant maize and triticale genotypes indicate that resistant have more efficient protection mechanisms against water loss, cell membrane status, photosynthesis and WUE. Therefore, further studies on physiological and metabolic processes in sensitive and resistant genotypes are necessary, particularly in hydraulic and chemical signaling, sink-source relations and the supply of water and carbon.