Maize hybrids chosen for the experiments differed in susceptibility to soil compaction stress on the basis of stress susceptibility index (SSI) which was calculated according to Fischer and Maurer (1978). For M and S treatments SSI for Ankora was 1.40 and 1.17 and for Tina 0.61 and 0.69, respectively. After 28 days of seedling growth under low (L + C) or severe (S + C) soil compaction and optimal soil water content the decrease of dry weight was higher in Ankora than in Tina. Moreover, differences of seedlings’ dry matter between L + W or S + W and L + D or S + D were markedly higher for Ankora than for Tina (results not shown).
Diurnal changes of water potential in soil (
S), root (ψ
R) and leaf (ψ
L) in two maize hybrids
Measurements of ψ
R and ψ
L were taken after 14 days of development under low (L) and severe (S) soil compaction with optimal soil water content (C—about 65 % FWC) and over the next 7 successive days (from 14th to 21st day) for seedlings grown in pots without watering (L + D, S + D) and for seedlings flooded in a container with water (L + W, S + W). Samples of leaf, root and soil were taken from 8 am to 8 pm at 3-h intervals in 3 replications (Figs. 1, 2).
During 7 days of seedlings growth under drought (L + D, S + D) or waterlogging (L + W, S + W), the stresses strongly affected diurnal changes of ψ
R and ψ
L and also ψ
S but only in drought stressed plants (Fig. 1). For seedlings subjected to drought the differences in ψ
S between hybrids were observed under both L + D and S + D. For sensitive hybrid (Ankora) in L + D and S + D treatments ψ
S decreased from −0.22 to −1.60 and from −0.31 to −1.75 MPa, respectively, and in resistant hybrid (Tina) from −0.18 to −1.20 MPa and from −0.21 to −1.51 MPa, respectively. During the successive days of seedlings’ growth without watering, differences between ψ
S and ψ
R and between ψ
S and ψ
L were observed and were highest around noon and later in the day (11 am, 2 and 5 pm) and lower in the morning (8 am) and evening (8 pm), particularly in the case of ψ
R. Also for L + D and S + D the differences between ψ
R and ψ
L around noon and later in the day in Ankora were small comparing to Tina (Fig. 1). In both soil compaction treatments and in waterlogging conditions the changes of ψ
R and ψ
L were lower in comparison with drought conditions (Fig. 2).
The reason for the changes in diurnal fluctuations of ψ
R and ψ
L under drought and waterlogging treatments around noon and in the afternoon is that the high rate of transpiration at midday is not counterbalanced completely by the roots’ water uptake from the soil. In the afternoon the evaporative demand gradually declines because more water enters the plant through the roots than is transpired by the leaves. The tissue again becomes filled with water, and ψ
L and ψ
R increase. At the end of the night an almost complete balance is achieved between ψ
L and ψ
S. This complete recovery is attained after the 1st, 2nd and 3rd day, but is no longer achieved between 4th and 7th day. Table 1 shows the analysis of variance (ANOVA) of ψ
R and ψ
L in the two maize hybrids grown under L + D, L + W, S + D and S + W treatments. For all factors (h—hybrids, t—treatments, c—soil compaction and d—day) and all interactions between the factors, the variance was statistically significant, with the exception of h × t × c in the leaf water potential.
Effects of drought (D) or waterlogging (W) stresses on membrane injury (LI), chlorophyll content (SPAD), leaf water potential (ψ
L) and leaf gas exchange parameters (P
N, E, g
Measurements of LI and SPAD were made in seedlings grown from sowing to 28th day in three soil compaction treatments (L, M, S) and under optimal soil water content (C) and under drought (D) or waterlogging (W) from 14th to 28th day. However, the measurements of ψ
N, E and g
S were carried out on seedlings grown only in L and S soil compaction and under C, D and W soil water content. The measurements were performed between 11 am and 1 pm.
Membrane injury index (LI)
After 28 days of seedling growth in M + D, M + W, S + D and S + W treatments the values of LI in the sensitive hybrid were larger than in the resistant one (Table 2). In M + C and S + C treatments the values of LI in Ankora were 8.1 and 11.1 and in Tina 6.4 and 8.5, respectively. Under drought (35 % FWC) in L + D, M + D and S + D treatments the values of LI in Ankora were 13.1, 18.3 and 29.4 and in Tina 11.3, 13.1 and 21.0, respectively. Under waterlogging stress (L + W, M + W, S + W) leaves were less leaky to solubles with LI values slightly lower than under drought—in Ankora 9.8, 13.0 and 18.5 and in Tina 6.9, 8.8 and 11.9, respectively.
Chlorophyll content (SPAD)
For seedlings grown under three soil compaction treatments and subjected to drought (L + D, M + D, S + D) or waterlogging (L + W, M + W, S + W), a decrease of SPAD was observed (Table 2). The decrease of SPAD in M + C and S + C treatments in comparison with L + C was about 25 and 58 %, respectively, in Ankora and 30 and 42 % in Tina. In low and moderate soil compaction (L + D, L + W, M + D and M + W) the differences between hybrids were small and often not statistically significant. Differences in SPAD between Ankora and Tina were observed both in S + D and S + W treatments. In comparison with L + C treatment, the decrease of SPAD in S + D and S + W treatments for Ankora was 30 and 31 %, respectively, and for Tina 23 and 21 %. The obtained results show that for the tolerant hybrid Tina grown under M + C or S + C the decrease of chlorophyll content was smaller in comparison with the sensitive hybrid Ankora.
Leaf water potential (ψ
L) and gas exchange parameters (P
N, E, g
The decrease of ψ
L in seedlings of both hybrids grown for 28 days under S + C conditions was small (about 10 %) in comparison with L + C (Table 3). In seedlings of both hybrids subjected to L + D or L + W the decrease of ψ
L was similar and at the same time it was about three times greater than in the case of L + C treatments. S + D treatment caused a decrease of ψ
L in Ankora by about 25 % and in Tina by about 7 % in comparison with L + D. Waterlogging stress caused a decrease of ψ
L in Ankora by about 20 % and in Tina by about 10 % (Table 3).
The decrease of P
N in seedlings grown under S + C treatment amounted to about 23 % in Ankora and 15 % in Tina but the differences in E and g
S between L + C and S + C were very small in both hybrids and amounted to about 29 % in the case of E and 14 % in the case of gS. The decrease of Pn in S + D and S + W treatments in comparison with L + D and L + W treatments was 27 % for Ankora and 22 % for Tina. Significant differences between both hybrids were observed in E and g
S. The decrease of E in S + D and S + W treatments in comparison with L + D and L + W treatments was 45 and 50 %, respectively, for Ankora and 32 and 35 % for Tina. Similarly, the decrease of g
S in S + D and S + W treatments in comparison with L + D and L + W treatments was 29 and 18 %, respectively, for Ankora and about 40 % in both treatments for Tina. The variance was significant for all variables (h, c, t) but not for the interaction between hybrids and soil compaction variables (h × c).
Content of ABA and antioxidants activity
ABA content under control (L + C) conditions ranged from ca. 1.3 (roots) to 2.4 (leaves and stem) nmol g−1 DW (Fig. 3). There were no statistically significant differences between the hybrids investigated. Severe soil compaction or drought as a single stress increased ABA level in the stem and leaves but only in the tolerant hybrid Tina. In the case of drought, this ABA increase in the stem and leaves was combined with its significant decrease in roots. Multistresses affecting roots (S + D and S + W) did not change their ABA level but they substantially increased ABA content in the stem and leaves of the tolerant hybrid Tina and in the leaves of the sensitive hybrid Ankora. The ANOVA of ABA content and all factors investigated (Table 4) shows statistically significant interaction between hybrids and soil compaction (h × c) for ABA content in the stem, which indicates hybrid-specific impact of soil compaction on stem ABA content. Similar interaction is observed between soil compaction and water availability.
Total antioxidant activity under control (L + C) conditions was manifold higher in leaves than in stem or root tissue and it differed significantly between hybrids only in the stem (Fig. 4). Under severe soil compaction it increased significantly in both hybrids but only in stem tissue. Multistress S + D substantially decreased antioxidant activity in the roots of both hybrids. The ANOVA of total antioxidant activity and all factors investigated (Table 5) shows hybrid-specific interaction with soil compaction and water availability in the case of antioxidant activity in the stem and leaf (h × c × t).
Non-enzymatic antioxidants play an important role in antioxidant defence system and are involved in redox signalling in plants under various environmental stresses. Any substantial changes of their activity can be interpreted as an oxidative stress. Significant decrease of their activity in roots under water shortage conditions (L + D, S + D) in comparison to control conditions (L + C) confirms that their biosynthesis rate is lower than their usage for scavenging free radicals. On the other hand, severe soil compaction (S + C) significantly increased their activity in the stem, whereas combined stress (L + D and S + W only in Tina) alleviated this effect (Fig. 4).
Correlation between the measured physiological markers
Statistically significant linear correlation coefficients (r) between ABA content and antioxidant activity were found only in root and stem for resistant hybrid Tina. For this hybrid a statistically significant correlation was also found between ABA content and antioxidant activity in root and ψ, P
N, E and g
S. For both hybrids a statistically significant correlation was found between ψ and P
N, E and g
N and E and g
S, and E and g
S. For soil compaction sensitive hybrid Ankora significant correlations were also found between LI and SPAD, ψ, P
N, E and g
S but for resistant hybrid Tina only between SPAD and E and gs (Table 6).
Interaction between physiological and biochemical markers According to Mittler (2006) the interactions between physiological and biochemical markers will be determined by comparison of separate effects of drought or waterlogging stresses (D, W) with their combined effects with soil compaction stress (L, S). This interaction may be described as (1) no interaction when differences are lower than (±10 %), (2) potentially negative interaction, in a situation where the combined effect of both stressors is higher (+10 %) than the effects of only one of them, and (3) potentially positive interaction in a situation where the combined effect of the two stressors is lower (10 %) than the effects of only one of them. The physiological markers used in this study (membrane injury, water potential, gas exchange parameters, chlorophyll content) indicated only potentially negative interaction between soil compaction and limited or excessive water content in soil. Potentially negative interactions in Ankora were always higher than in Tina as well as in seedlings grown under low or severe soil compaction. In the case of biochemical markers (ABA content and antioxidant activity) the nature of the interaction between L or S soil compaction and soil water content (D, W) was not clear (Table 7). In Ankora potentially negative interactions for ABA content were observed in six cases and for antioxidant activity in five cases and in Tina in three and four cases, respectively. In other cases, both in Ankora and Tina the interactions were potentially positive or no interactions were found.