-
Under control conditions, no significant differences were observed in phenotype among maize lines (Fig. 1a). The differences among maize lines in leaf phenotype occurred under SS and after RSS (Table 1; Fig. 1a). Under SS, Z58 showed no significant changes in leaf phenotype, and more than 95% of C7-2 leaves died after SS of 72 h (Table 1; Fig. 1a). After RSS, all seedlings of 72 h-SS-stressed Z58 survived, however, all seedlings of 72 h-SS-stressed C7-2 died (Table 1; Fig. 1a).
Figure 1.
(a) Phenotype, (b) leaf RWC, and (c) root staining of maize inbred lines under SS and after RSS. The SS stress was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. In (b), the data were the means ± standard deviation (SD) of the fully expanded 2nd leaves of 5-leaf-old seedlings (n = 5−10) for each maize line under each treatment, and statistical analysis comparison was conducted between the same maize lines under control and the same SS stress, and between the same maize lines after SS of 72 h and after RSS. In (b), upper and lower cases of the same letter indicated a statistical significance at p < 0.05. In (c), fresh nodal roots (1 cm behind the root tip) from 5-leaf-old seedlings (n = 5−10) of each maize line were stained with Evans blue solution. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. RWC, Relative water content. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
Table 1. Phenotypes of seedlings of maize inbred lines under SS and after RSS.
Maize line Yellowing of leaves Death rate (%) of seedlings under SS for After RSS 5 h 24 h 48 h 72 h Surviving seedlings (%) Z58 Slightly; The edge of about 10% of leaves after SS of 72 h 0 0 0 0 100 Y478 Obviously; About 15% of leaves began turning yellow after SS of 48 h 0 0 1.2 2.1 50.5 C7-2 Obviously; About 15% of the leaves began turning yellow after SS of 24 h 0 0 40.5 95 0 HZ4 Somewhat like C7-2 0 0 23.5 80 48.65 The SS stress was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. Leaves were observed and counted from leaves of 15−20 seedlings for each maize line. Survival rate after RSS referred to the percentag of surviving seedlings compared to seedlings stressed after SS of 72 h. C7-2, Maize inbred line Chang 7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58. Leaf RWC started to significantly decrease after SS of 5 h but had no significant difference among maize lines. The significant differences of maize lines in leaf RWC occurred after SS of 24, 48 and 72 h, respectively. Leaf RWC was highest in Z58 and lowest in C7-2 after SS of 72 h (Fig. 1b). Notably, leaf RWC of Z58 did not significantly fluctuate under SS (Fig. 1b).
However, leaf RWC of Z58, Y478 and HZ4 significantly increased after RSS when compared to that in their respective maize lines SS-stressed for 72 h but it was still significantly lower than that of their respective control lines (Fig. 1b).
The deeper the Evans blue staining indicated the less viability of cells. Consequently, the visible staining differences occurred among roots of maize lines after SS of 5 h and more significantly after SS of 24 h (Fig. 1c), where Evans blue-stained root zone was close to root tips of SS-stressed Z58 but relatively longer in SS-stressed lines of Y478, HZ4 and C7-2 (Fig. 1c).
After RSS, Evans blue-staining was only at local root zone in SS-stressed Z58 but still in a longer root zone in SS-stressed lines of Y478 and HZ4. The staining depth of SS-stressed roots of maize lines followed Z58 < Y478 < HZ4 (Fig. 1c) but was shallower than that of their respective maize lines after SS of 72 h. Such staining differences were echoed partly by the quantitative assay of Evans blue (Fig. 2a).
Figure 2.
Evans blue content in (a) fresh nodal roots, and Na+ content in (b) roots and (c) leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. In (a), Evans blue content analysis was based on the Evans blue-stained fresh nodal roots (1 cm behind the root tip), where each datum was the mean ± SD from 5-leaf-old seedlings (n = 3−5) for each maize line under each treatment. In (b), each datum was the mean ± SD from a collection of roots of 5-leaf-old seedlings (n = 5−10). In (c), each datum was the mean ± SD from the fully expanded 2nd leaves of 5-leaf-old seedlings (n = 5−10). The statistical analysis comparison was conducted between the same maize line under control and the same SS stress, and between the same maize line after SS of 72 h and after RSS. The upper and lower cases of the same letter indicated a statistical significance at p < 0.05. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SD, Standard deviation. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
Both phenotype of shoots (Table 1; Fig. 1a) and root staining (Fig. 1c) under SS and after RSS indicated that SS tolerance degree of maize lines roughly followed Z58 > Y478 > HZ4 > C7-2.
Na+ content in the tissues
-
Under control conditions, there were very slight but no significant differences in Na+ content of either roots (Fig. 2b) or leaves (Fig. 2c) among different maize lines.
Na+ content in roots of SS-stressed maize lines significantly increased when compared to that in their respective control-treated lines, which roughly fluctuated as follows: highest in roots of Z58 after SS of 5 and 24 h, and no significant differences among maize lines after SS of 48 and 72 h (Fig. 2b). After RSS, Na+ content in roots of SS-stressed maize lines of Z58, Y478 and HZ4 significantly decreased when compared to that in their respective maize lines after SS of 72 h but it was still significantly higher than that in their respective control lines (Fig. 2a). It should be noted that after RSS Na+ content in roots of SS-stressed Z58 was still highest among SS-stressed maize lines, similar to the situation in its roots after SS of 5 and 24 h (Fig. 2b).
Na+ content in leaves of SS-stressed C7-2 was highest among SS-stressed maize lines (Fig. 2c). After RSS (Fig. 2c), changes in Na+ content in leaves of SS-stressed maize lines were very similar to those in roots (Fig. 2b) of SS-stressed maize lines. Overall, the absolute Na+ content was much higher in leaves than in roots for each SS-stressed maize line.
K+ and Ca2+ contents in the tissues
-
Overall, K+ content in roots (Fig. 3a) and leaves (Fig. 3b) of Z58 was highest among maize lines under either control conditions or SS stress.
Figure 3.
K+ content in (a) roots and (b) leaves, and Ca2+ content in (c) roots and (d) leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. Each datum was the mean ± SD from the fully expanded 2nd leaves or a collection of roots of 5-leaf-old seedlings (n = 3−5) for each maize line under each treatment. The statistical analysis comparison was conducted between the same maize line under control and the same SS stress, and between the same maize line after SS of 72 h and after RSS. The upper and lower cases of the same letter indicated a statistical significance at p < 0.05. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SD, Standard deviation. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
As for K+ content in roots of SS-stressed maize lines, it showed no significant changes in Z58, significantly decreased in Y478 and HZ4, and significantly increased in C7-2 after SS of 5 h. K+ content tended to decrease although it fluctuated in some maize lines after SS of 24 h (Fig. 3a). After RSS, K+ content in SS-stressed maize lines of Z58 and Y478 was still lower than that in their respective control lines (Fig. 3a).
Regarding K+ content in leaves of SS-stressed maize lines, it showed no change in Z58, significant decreases in Y478 and C7-2 and a significant increase in HZ4 after SS of 5 h when compared to that in their respective control lines. However, as the SS time prolonged, although K+ content was significantly lower than their respective control maize lines, it fluctuated obviously with maize lines. In general, K+ content in roots (Fig. 3a) and leaves (Fig. 3b) of C7-2 after SS of 48 and 72 h was lowest among SS-stressed maize lines. After RSS, K+ content in SS-stressed lines of Z58 and HZ4 was very close that in their respective control lines (Fig. 3b).
With aspect to Ca2+ content in roots of SS-stressed maize lines, it significantly increased in Z58 and C7-2, and significantly decreased in Y478 and HZ4 after SS of 5 h (Fig. 3c). Ca2+ content in Z58 significantly decreased but remained relatively constant as SS exceeded 5 h. After RSS, Ca2+ content was still lower in SS-stressed Z58, sharply increased in SS-stressed Y478, and recovered to the control level in SS-stressed HZ4 when compared to that in their respective control maize lines (Fig. 3c).
As regards Ca2+ content in leaves, it was highest in HZ4 and lowest in Z58 under control conditions. Interestingly, as for Ca2+ content under SS, it changed greatly with maize lines, either increased or decreased at some SS-time points. however, it was still lowest in SS-stressed Z58 when compred to that in teir respective control lines (Fig. 3d). After RSS, Ca2+ content significantly increased in SS-stressed lines of Z58 and Y478 (Fig. 3d).
Malondialdehyde content in the tissues
-
In general, malondialdehyde content in roots (Fig. 4a) and leaves (Fig. 4b) of all SS-stressed maize lines tended to significantly increase as SS time prolonged when compared to that in their respective control maize lines, highest in roots (Fig. 4a) and higher in the most cases in leaves (Fig. 4b) of SS-stressed lines of HZ4 and C7-2. Notably, malondialdehyde content in roots (Fig. 4a) and (Fig. 4b) of SS-stressed Z58 and Y478 showed slight changes when SS time was over 24 h, not as dramatically increased as in other SS-stressed maize lines (Fig. 4a). After RSS, malondialdehyde content in roots (Fig. 4a) and leaves (Fig. 4b) of SS-stressed maize lines significantly decreased when compared to that in their respective lines that were SS-stressed for 72 h, but it was still higher than that in their respective control maize lines, highest in SS-stressed HZ4.
Figure 4.
Malondialdehyde content in (a) roots and (b) leaves, and SAR content in (c) roots and (d) leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. Each datum was the mean ± SD from the fully expanded 2nd leaves or a collection of the roots of 5-leaf-old seedlings (n = 3−5) for each maize line. The statistical analysis comparison was conducted between the same maize line under control and the same SS stress, and between the same maize line after SS of 72 h and after RSS. The upper and lower cases of the same letter indicated a statistical significance at p < 0.05. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SAR, Superoxide anion radical. SD, Standard deviation. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
SAR production in the tissues
-
As for SAR production in roots, no significant differences were found among maize lines under control conditions (Fig. 4a). Under SS stress, SAR production significantly increased in maize lines but was highest in Z58 especially after SS of 5 h. However, SAR production tended to significantly decline in SS-stressed maize lines when SS time was at and over 24 h although it was higher than that in their respective control lines. After RSS, SAR production situation in SS-stressed lines of Z58, Y478 and HZ4 was almost the same as that in their respective maize lines that were SS-stressed for 72 h (Fig. 4c).
Regarding SAR production in leaves, SAR production in Z58 and Y478 was much higher than that in HZ4 and C7-2 under control conditions (Fig. 4d). Under SS stress, SAR production was always much lower in most SS-stressed maize lines after SS of 5, 24, and 48 h, and significantly decreased in Y478 but increased in HZ4 and C7-2 after SS of 72 h (Fig. 4d) when compared to that in their respective control lines. After SS, SAR production was almost identical to that in their respective maize lines that were SS-stressed for 72 h (Fig. 4d).
Activities of SOD and POD in the tissues
-
As for SOD activity in roots, it showed differences among maize lines under control conditions, and significantly increased but was highest in Z58 under SS (Fig. 5a). For SOD activity in leaves, it was much higher in Z58 and Y478 under control conditions, and significantly decreased in Z58, Y478 and C7-2 but significantly increased in HZ4 after SS of 48 and 72 h (Fig. 5b) when compared to that in their respective control lines. After SS, SOD activity was higher in roots of SS-stressed maize lines (Fig. 5a) and only in leaves of SS-stressed HZ4 when compared to that in their respective control lines (Fig. 5b).
Figure 5.
SOD activity in (a) roots and (b) leaves, and POD activity in (c) roots and (d) leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. Each datum was the mean ± SD from the fully expanded 2nd leaves or a collection of the roots of 5-leaf-old seedlings (n = 3−5) for each maize line under each treatment. The statistical analysis comparison was conducted between the same maize line under control and the same SS stress, and between the same maize line after SS of 72 h and after RSS. The upper and lower cases of the same letter indicated a statistical significance at p < 0.05. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. POD, Peroxidase. RSS, Removal of SS. SD, Standard deviation. SOD, Superoxide dismutase. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
Overall, the changing patterns of POD activity in either roots (Fig. 5c) or leaves (Fig. 5d) of maize lines either under control conditions and SS or after RSS were almost in line with those SOD activity in corresponding roots (Fig. 5a) or leaves (Fig. 5b) .
Activities of CAT and APX in the tissues
-
As for CAT activity in roots under SS, it significantly increased in all SS-stressed maize lines (Fig. 6a) but was much higher in SS-stressed Z58 than that in other SS-stressed maize lines especially after SS of 5 h. In leaves under SS, overall, CAT activity significantly increased after SS of 5, and 24 h for all SS-stressed maize lines and significantly decreased after SS of 48, and 72 h for SS-stressed maize lines of Z58 and Y478 (Fig. 6b). Notably, CAT activity was always higher in leaves of SS-stresed maize lines of HZ4 and C7-2 than that in their respective control lines (Fig. 6a). After SS, it was higher in roots (Fig. 6a) of all SS-stressed maize lines, and higher in leaves (Fig. 6b) of SS-stressed maize lines of Z58 and HZ4 than that in their respective control lines.
Figure 6.
CAT activity in (a) roots and (b) leaves, and APX activity in (c) roots and (d) leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. Each datum was the mean ± SD from the fully expanded 2nd leaves or a collection of the roots of 5-leaf-old seedlings (n = 3−5) for each maize line under each treatment. The statistical analysis comparison was conducted between the same maize line under control and the same SS stress, and between the same maize line after SS of 72 h and after RSS. The upper and lower cases of the same letter indicated a statistical significance at p < 0.05. The data were not available for maize inbred line C7-2 after RSS because of no surviving seedlings. APX, Ascorbate peroxidase; CAT, Catalase. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SD, Standard deviation. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
Overall, APX activity changes in roots (Fig. 6c) under control conditions and SS or after RSS were somewhat similar to CAT activity found in roots (Fig. 6a) although there were slight differences for some maize lines. As for APX activity in leaves, it was always higher in SS-stressed maize lines of HZ4 and C7-2, but significantly lower in SS-stressed maize lines of Z58 and Y478 after SS of 72 h when compared to that in their respective control lines (Fig. 6d). After RSS, APX activity was much higher in roots (Fig. 6c) of SS-stressed maize lines of Z58 and HZ4, and lower in leaves (Fig. 6d) of SS-stressed maize lines of Z58 and Y478 but much higher in leaves (Fig. 6d) of SS-stressed HZ4 when compared to that in their respective control lines.
Stomatal behaviour of leaves
-
The leaf stomata were always opened in Z58 under SS, began to close in HZ4 and C7-2 after SS of 24 h and in Y478 after SS of 48 h (Fig. 7a) when compared to those of their respective controls lines (Fig. 7b). After RSS, the leaf stomata were still opened in SS-stressed Z58, slightly opened in SS-stressed Y478, and still closed in SS-stressed HZ4 (Fig. 7a).
Figure 7.
Stomatal behaviour in leaves of maize inbred lines under SS and after RSS. The SS was conducted with 150 mM NaCl. The RSS treatment was performed on maize plants stressed by SS for 72 h. The photos of the leaf stomata were taken by SEM from the central region of the front surface of the fully expanded 2nd leaves of 3-leaf-old seedlings (n = 5) for each maize line under each treatment. The data of maize inbred line C7-2 after RSS were not available because of no surviving seedlings. C7-2, Maize inbred line Chang7-2. HZ4, Maize inbred line Huangzao4. RSS, Removal of SS. SEM, Scanning electron microscopy. SS, Salt shock. Y478, Maize inbred line Ye478. Z58, Maize inbred line Zheng58.
-
We are grateful to Professors Yu Li and Yun-Su Shi, the Institute of Crop Sciences, CAAS, who kindly supplied the maize seeds.
-
About this article
Cite this article
Pan JL, Fan XW, Li YZ. 2023. Insights into physio-biochemical responses of maize to salt shock stress and removal of the stress at the whole-plant level. Tropical Plants 2:20 doi: 10.48130/TP-2023-0020
Insights into physio-biochemical responses of maize to salt shock stress and removal of the stress at the whole-plant level
- Received: 28 April 2023
- Accepted: 17 October 2023
- Published online: 07 November 2023
Abstract: Maize (Zea mays) is one of the world's three major food crops but sensitive to salinity at the seedling stage. Salinity/salt stress usually occurs due to gradually increased NaCl or under sudden exposure to NaCl, of which the latter is called salt shock (SS). However, little is known about physio-biochemical responses of maize to SS at the whole-plant level. The purpose of this study was to characterize the physio-biochemical response events of maize under SS. The experiments were conducted with four maize foundation parent inbred lines of Huangzao4, Chang7-2, Zheng58 and Ye478 under 150 mM NaCl for SS and after removal of SS in the nutrient solutions. The main findings were that the maize lines had no clear phase-order-response to SS, which suffered from the combined effects of osmotic stress, water deficiency, and Na+ accumulation-induced toxicity once SS occurred, and that SS-tolerant maize lines showed (1) timely increased activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) and stronger superoxide anion radical-mediated signalling in roots at the beginning of SS, (2) a slow Na+ transport rate from roots to shoots especially in the early SS stage, and (3) opening of leaf stomata, and fine cell membrane integrity during SS. The related mechanisms of SS tolerance of maize were proposed and discussed.
-
Key words:
- Maize /
- Salt shock response /
- Antioxidant enzymes /
- Ca and K ions /
- Leaf stomata