Growth, photosynthesis parameters and pigment content
During their seedling stage, plants are sensitive to adverse external factors; therefore, seedlings stage is the optimum time to research plants abiotic tolerance [33]. RGR could reflect the growth conditions of a plant and is considered as an important index in determining the degree of stress of plants. Salinity generally inhibits plants growth and even leads to death [15]. Our findings evidenced the adverse effect of alkaline salt stress on root growth (Fig. 1a and b). These observations indicated that although the impacts of neutral salt and alkaline salt stresses are similar to a certain extent, they are actually two distinct kinds of stresses. The additional impact of high-pH stress under high pH conditions contributes to achieving even more pronounced harmful effects than the ones caused by salinity stress [15, 16].
To obtain insights into the mechanisms involved and the nature of stress-induced damage to the photosynthetic apparatus, we also examined the changes in the photosynthesis and pigment content, which are parameters of stress as reported earlier [16]. Moderate levels of neutral salt stress had a little impact on the major parameters of photosynthesis, whereas alkaline salt stress exerted a more severe adverse influence, leading to a decline in P
n, g
s and E (Fig. 1c–e, P < 0.01). In addition, the chlorophyll content was not diminished in the neutral salt stress treatment, but it declined sharply under the conditions of alkaline salt stress (Fig. 1f, P < 0.01). It is well known that plant species have three metabolism processes response to massive Na+ under salt stress, including exclusion, compartmentalization and ion transport [34]. The Na+ exclusion mechanism dependent on a Na+/H+ antiport, such as salt overly sensitive 1 type (SOS1), and the transmembrane proton gradient (H+-ATPase) decided to exchange activity of Na+ and H+ [23]. The high pH value decreased external protons and weakens the exchange activity of the Na+/H+ antiport, causing the exclusion of Na+ has been inhibited and enhancing Na+ accumulated in vivo under alkaline salt stress [34, 35]. The negative action of alkaline salt stress on photosynthetic capacity and chlorophyll content was probably due to the accumulation of Na+ in the cytoplasm as well as to the destruction of the structure and suppression of the functions of chloroplasts [16, 35]. Superfluous Na+ and high pH value affect Fe accumulation; which is known to play important role in chlorophyll biosynthesis and photosynthetic rate in plants. The content of Fe decreased caused great decrease in content of chlorophyll content, photosynthesis and therefore a decreased in biomass [36–38].
Metal elements
The cytoplasm of higher plants normally maintains high K+ and low Na+ concentrations to facilitate the proper functioning of many enzymes and the normal action of the catalyzed by them important physiological processes; osmotic regulation is the main mechanism to sustain this state [1, 23]. The findings of this investigation confirm that competitive relationships exist between K and Na during their uptake under the conditions of high salt and alkalinity stress; the amount of Na increased, while the total K content decreased. These effects were more pronounced under alkaline salt stress than under neutral salt stress. Maize plants respond to the stress caused by high pH by a considerable increase in the accumulation of Na and Ca in their tissues, a reaction that does not occur under neutral salt stress. A large number of plants possess a remarkable mechanism for exclusion of Na+ that is dependent on the gradient of H+ across the cell membranes of the roots [34]. For example, in the model plant Arabidopsis, SOS1 protein has been identified that it functions in exclusion of Na+ from epidermal cells of roots to the rhizosphere, which may play an important role in retrieving Na+ from roots to shoots under salt stress, so this phenomenon might be the basis metabolism response to alkaline injury [34]. In addition, the research found that Ca2+ plays important roles in the regulating AtSOS3–AtSOS2 protein kinase pathway mediates expression, and it also responsive AtNHX and AtSOS1 protein regulation activities of Na+ transporters, which indicated that Ca2+ being the key signal component in the SOS system in Arabidopsis and some other plant species [23, 34]. In conclusion, we infer that by excluding Na+ and Ca2+ play important roles in plant alkaline tolerance. In this study, neutral salt stress reduced Ca2+ accumulation in maize roots, but alkaline salt stress strongly enhanced its accumulation in the shoots and roots. The increase of Ca2+ level in tissues of maize seedlings during alkaline salt stress can instantly activate the SOS–Na+ system for exclusion and diminish the damage to the plants caused by Na+ toxicity.
Neutral salt and alkaline salt stresses responses in maize metabolism
The excessive concentration of Na+ and the osmotic stress caused by high salinity have adverse impacts on the functions of the roots, inducing the generation of reactive oxygen species, such as H2O2 and O2
3 −, and causing intracellular hyper-ammonia stress [34, 39]. Under saline conditions, to decrease the water potential of the cytoplasm to prevent it from dehydration, plants usually accumulate organic solutes in their vacuoles, such as betaine, proline, free sugars, and polyalcohol [11, 34].
The results indicated that the GC-MS metabonomic analysis is an excellent method for understanding the molecular responses to salinity, which could reflect the integration of genomics, proteomics, transcriptomics and other different regulatory processes [40]. By protecting plant cell membranes and proteins and by functioning as a scavenger of reactive oxygen species, proline plays an important role in the response of plants to neutral salt stress [41, 42]. In the present examination, we detected dramatically elevated levels of proline in both the roots and shoots, which contributed significantly to the osmotic regulation in the experimental maize plants subjected to neutral salt stress. Similar findings on this protective function of proline were obtained by Wu et al. [43] and Yang et al. [16]. The neutral salt stress-induced elevation of glutamate levels indicates that proline biosynthesis is important for the control of salinity-induced osmotic pressure. However, the level of proline accumulation was significantly lower under alkaline salt stress than under neutral salt stress. Our results imply that the high pH values under alkaline salt stress conditions might suppress the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS), inhibiting the conversion of glutamate into proline.
The concentrations of sugars, such as glucose, fructose, and sucrose, have been found to increase in response to neutral salt stress [16, 44]. Our results showed that the levels of fructose, sucrose, talose, and myo-inositol in the roots, as well as those of raffinose and galactinol in the shoots, were dramatically increased in the maize plants under neutral salt stress, but glucose showed decreased trend (Table 3). In Gavaghan et al. [45] study, it confirmed that sucrose was increased significantly while glucose decreased in roots of maize under salt stress using nuclear magnetic resonance (NMR) spectroscopy. In plants, sugars are commonly produced by photosynthesis, degradation of polysaccharides, and gluconeogenesis [44]. In our investigation, we found that the photosynthetic rate of the seedlings subjected to neutral salt stress was similar to that of the ones in the control group. This result suggests that the process of gluconeogenesis was enhanced in the plants under neutral salt stress, implying that degradation of polysaccharides, used as a carbon source, was probably promoted to achieve maintenance of osmotic balance (Fig. 1 and Table 3). Nevertheless, the concentrations of glucose, sucrose, and ribose were significantly reduced in the experimental maize seedlings in response to alkaline salt stress. The rate of photosynthesis was substantially decreased by alkaline salt stress, resulting in inhibited production of reducing forces and limited N metabolism, which in turn lowered sugar production (Fig. 1 and Table 3). The toxic levels of Na+ that had accumulated in plant cells at high pH values might have also had detrimental effects on sugar production.
In the present study, evident differences between the responses to neutral salt and alkaline salt stresses were found in the content of metabolites in the investigated maize plants. Neutral salt stress stimulated sugar accumulation, but glycolysis, the shikimic pathway, and amino acid synthesis in roots, were inhibited (Fig. 3). By contrast, glycolysis and the synthesis of amino acids and fatty acids in shoots were enhanced, while the TCA cycle was suppressed (Fig. 4). These results indicate that under neutral salt stress, the most important compatible solutes are the sugars in the roots and that active synthesis metabolism is a basic response of shoots in developing their tolerance to neutral salt stress. The increased levels of serine, isoleucine, and phenylalanine in shoots were probably related to glycolysis as a way of relieving transamination products because they are glucogenic amino acids (Fig. 3). Fatty acids maybe an important compatible solute in shoots of maize plants subjected to neutral salt stress, especially palmitic acid and oleic acid
Under alkaline salt stress, the TCA cycle, shikimic pathway, and organic accumulation were enhanced significantly; however, the synthesis of amino acids was inhibited significantly in the roots. Furthermore, a decrease in the content of glutamate and alanine indicated that the accumulation of these amino acids enhanced GABA shut biosynthesis process, leading to increases in the TCA cycle (Fig. 3). Under alkaline salt stress, the glycolysis and synthesis of amino acids and fatty acids in shoots were inhibited (Fig. 4). These results indicated that energy and high levels of organic acids are the key adaptive mechanisms by which maize seedlings maintain their intracellular ion homeostasis under alkaline salt stress. The accumulation of organic acids in vacuoles might play a central role in the regulation of intracellular pH through neutralization of excess cations [15, 16]. Excessive Na+ ion concentrations may induce a cascade of signal transduction events which culminate in the promotion of the synthesis of organic acid leading to the negative charge deficit in maize. Consequently, accumulation of various organic acids in plant cells is necessary.
The reduction in amino acid levels in maize tissues induced by high pH could be attributed to the decrease of N metabolism rates. To realize absorption of nutrients, such as nitrates (NO3
−) and ammonium (NH4
+), the roots of plants utilize a number of transport systems [17]. For instance, the members of AMT protein family perform transportation of NH4
+, while the representatives of the NRT protein family realize the transport of NO3
−. NRT regulates the uptake of NO3
−, whereas AMT controls the absorption of NH4
+ possibly through the transmembrane proton gradient [46]. The absence of an external for the plasma membrane of the roots supply of protons under alkaline salt stress conditions might retard the actions of NRT and AMT, leading to a decrease in the uptake of NO3
− and NH4
+. This phenomenon might influence nearly all processes of plant metabolism, which was confirmed by our findings. At high pH values, alkaline salt stress considerably suppressed the rate of photosynthesis, leading to a decline in glycolysis, reduced production of sugars and amino acid, and limited N metabolism. Consequently, we speculate that high concentrations of organic acids and energy are potential major factors whose action is required for the adaptation of maize plants, achieving proper support of the balance of intracellular ion concentrations and exerting control on high pH values under high alkaline salt stress.