The tested material in this experiment is the hybridized maize (Zea mays L.) cultivar called “zhengdan958”. According to [48–51], the fully-nourished seeds were selected, sterilized by 0.1% HgCl₂ for 10 min and repeatedly washed by water. 24 h after seed soaking, the seeds were cultured in an incubator for germination with a constant temperature of 28 °C. When the bud length reached 1 cm, the seeds were planted into vermiculite pots. When the first true leaf was entirely extended, the seedlings were sampled. The sample leaves were quickly soaked into PEG-6000 (−0.85 MPa) for stress treatments of 30, 45, 60, and 75 min, respectively. After these treatments, cytochemical localization was implemented in the cells of sample leaves. The sample leaves were then collected for the following treatments: (1) treated by the direct addition of 50 μM ABA; (2) soaked into 2 mM EGTA solution (supplemented with 10 mM MES, 50 mM KC1, pH 6.1) for 30 min for illumination avoidance and then treated by 50 μM ABA; (3) soaked into 100 μM Verapamil solution (supplemented with 10 mM MES, 50 mM KC1, pH 6.1) for 30 min for illumination avoidance and then treated by 50 μM ABA; (4) soaked into 80 μM TFP solution (supplemented with 10 mM MES, 50 mM KC1, pH 6.1) for 30 min for illumination avoidance and then treated by 50 μM ABA. The cytochemical localization of Ca²⁺ was implemented as following: 2% potassium pyroantinonate solution (represented by “A solution” in the following context, pH 7.6) was formulated with potassium phosphate. A solution should be formulated as a fixed solution containing 3% glutaraldehyde. The sample leaves were treated by such reagent with a size of 1 mm × 0.3 mm × 10 mm and fixed overnight. After overnight, theses leaves were washed by A solution for 3 times with an interval of 0.5 h for each time. Then, theses samples were fixed again by osmic acid formulated with A solution for 2 h, washed 3 times by double distilled water with an interval of 0.5 h for each time, and then washed twice by double distilled water (pH 10) with an interval of 0.5 h for each time. Next, the samples were placed into ethanol with various concentration gradients, and dehydrated, embedded, ultramicrocut, and dyed with uranium acetate, respectively. After the above operations, the approximate configuration and dense electronic particles of these leaves can be observed under transmission electron microscope (TEM), thus further inferring the location of Ca²⁺. The Ca²⁺ cellular localization was determined as follows: the photographed slices were placed into EGTA (a kind of Ca²⁺ chelators) for their chelating with Ca²⁺, the above slices were photographed when the dense electronic particles disappeared, and then the Ca²⁺ cellular distribution was determined.

It can be concluded from the results of TEM observation that calcium pyroantinonate precipitate particles created from cellular Ca²⁺ localization appeared in large quantity in vacuoles, and occurred occasionally in chloroplasts, nucleus and Golgi apparatus. It was reported that Ca²⁺ distributed in endoplasmic reticulum (ER) which also represents a commonly-considered cellular Ca²⁺ sink with large quantity, while such large amounts of Ca²⁺ distribution was not found in ER in this experiment. Plant cell wall represents another kind of cellular Ca²⁺ sink, and in this experiment, Ca²⁺ existence was found in cell walls. In addition, large amounts of calcium pyroantinonate precipitate particles were also observed in the intercellular gaps outside the cells (CK).

In Fig. 1.1, it can be observed that 30 min after PEG stress, the free Ca²⁺ concentration in cytoplasm and nucleus considerably increased. The large amounts of Ca²⁺ precipitate particles formed in the internal sides of the plasma membrane represented remarkable marks for such cellular Ca²⁺ concentration elevation. These particles were relatively orderly arranged and almost formed a particle circle in the internal sides of the plasma membrane. In vacuoles, Ca²⁺ was localized in the outside of vacuolar membrane. After PEG stress, vacuolar Ca²⁺ concentration significantly decreased (Fig. 1.1a), and large amounts of Ca²⁺ distribution occurred in the outside of chloroplast capsules (Fig. 1.1a). Contrary to the apparent change in cellular Ca²⁺ concentration, no remarkable change in cellular super-microstructure was observed (Fig. 1.1b). As shown in Fig. 1.2a, 45 min after PEG stress, the cells transformed, but such cell transformation was not significant. Relatively larger Ca²⁺ precipitates can be observed in the outside of cell walls, and relatively huge amounts of Ca²⁺ precipitate particles can also be found in cytoplasm. Compared with those under 30 min PEG stress treatment, the precipitates under this treatment were disorderly and unevenly distributed, and considerably concentrated in certain parts of plant cells. Some chloroplasts expanded and became round with their enlarged internal stromal spaces and expanded granular thylakoid, while the morphological change occurred in some other chloroplasts (Fig. 1.2a). With the continuous increase of Ca²⁺ concentration in the nucleuses and the appearance of larger black particles, the heterochromatin in the cells conglomerated (Fig. 1.2b). With cell transformation, the cytosolic Ca²⁺ concentration enhanced significantly, while the Ca²⁺ particles had irregularity in their configurations, some of them were big and dense black particles, while some others of them presented irregular hollow formations (Fig. 1.3a). In chloroplasts, Ca²⁺ concentration improved dramatically, and with the further expansion of granular thylakoid membranes, the distorted parts of membranes and the lamellar structures become vague while observed, and the increase of osmic acidophilic particle numbers took place in some chloroplasts (Fig. 1.3b). Some chloroplasts conglomerated with each other, forming a “butterfly configuration” (Fig. 1.3c). With relatively large amounts of Ca²⁺ particles mainly concentrated in the centre of nucleus, chromatins distributed in all directions around the nucleus (Fig. 1.3d). Observing Fig. 1.4a, it can be seen that cytosolic Ca²⁺ assembled in large quantities in certain cellular areas, and the occurrence of membrane breakages was found in some Ca²⁺ precipitate-centralized cellular areas. The serious chloroplast transformation was presented, and the black block masses with high electron density appeared after the partial breakage of some chloroplasts and the exosmosis of their stroma (Fig. 1.4b). With its collapsed and sectional vague membranes, the nucleus suffered from its serious transformation (Fig. 1.4c).

According to Fig. 2.1, it can be known that cellular after the ABA treatment to seedling leaves, cellular Ca²⁺ distribution changed significantly. The concentrations of Ca²⁺ precipitates decreased both in vacuoles and outside cells, while huge amounts of Ca²⁺ precipitate particles can be observed in the internal sides of membranes with the dramatic enhancement of cytosolic Ca²⁺ concentration. Compared with ABA treatment, EGTA and ABA jointed treatment had quite different effects on the changes of cellular Ca²⁺ distribution in seedling leaves. From Fig. 2.3, it can be observed that cellular Ca²⁺ concentration increased slightly, while Ca²⁺ outside cells can not be seen because of the elimination of it caused by EGTA chelating (Fig. 2.2). After Verapamil (an inhibitor to Ca²⁺ channels) and ABA jointed treatment, the cellular Ca²⁺ accession from outside into inside was inhibited, because Verapamil interrupted the Ca²⁺ channels in the membranes, thus leading to the obviously-observed large quantities of Ca²⁺ distribution in both intercellular gaps and cell walls. While the cytosolic Ca²⁺ concentration did not increase significantly (Fig. 2.3). TFP (an inhibitor to CAM) affected the cellular Ca²⁺ accession from outside into inside to some extent. Many Ca²⁺ precipitates can be seen in intercellular gaps, while the trend of cytosolic Ca²⁺ concentration elevation was not significant than such trend observed under ABA treatment (Fig. 2.4). After EGTA treatment, the precipitates in the slice were eliminated. The original sectors for Ca²⁺ distribution such as intercellular gaps, chloroplasts, vacuoles, and plasma membranes presented transparent electron areas similar as the original precipitate structures, demonstrating a real reflection of Ca²⁺ localization in the slice (Fig. 2.5).

The relationship between the nucleus of wheat seedling and Ca²⁺ under drought stress was examined with the cytochemical method of improved calcium antimonate precipitation. The results showed that there was a small concentration of Ca²⁺ in nucleus, the shape of the nucleus liked a ball and the chromatin was scattered distributed. The longer the duration of drought stress the higher the free Ca²⁺ concentration in nucleus and the more serious the ultrastructure of the nucleus, meanwhile, the membrane of the nucleus wrinkled and the chromatin seriously agglutinated, the shape of the nucleus was abnormal till its final disintegration [51–53]. It can be directly observed that under drought stress, the Ca²⁺ distributions and the cellular microstructures all changed in seedling leaves. At the early stage of drought stress, the nucleus and the chloroplast played important roles in regulating cellular Ca²⁺ concentrations, and the sectional distributions of Ca²⁺ in plant cells also provided vital roles in regulating the physiological activities regarding plant drought-resistance. Ca²⁺ participated in the processes of drought-stress and ABA-induced drought signal transduction as a pivotal medium. ABA could induce cytosolic Ca²⁺ concentration elevations in the mesophyll cells and guard cells of maize seedling leaves, such elevations originated mainly from extracellular Ca²⁺ sinks and partially from cellular Ca²⁺ sinks such as vacuoles. Extracellular CaM could participate in the process of drought signal transduction as an important medium through inducing the changes of cellular Ca²⁺ concentrations. It is also indicated and demonstrated in this experiment that under normal conditions, cellular free Ca²⁺ concentrations actually remain very low, and vacuole serves as a main Ca²⁺ sink for higher plants. Identical to the results of chemical measurement, this experiment also proved that there were relatively large quantities of Ca²⁺ distributions in cell walls and among intercellular gaps.

It has been proved by a growing number of researches that the sectional distribution of Ca²⁺ in cells can be changed through altering the transportation of Ca²⁺ carriers in membranes and the status of cellular Ca²⁺ storage bodies [5–9,21–28,45–49,53–56], which can be stimulated by many exogenous and endogenous elements such as light, low and high temperatures, gravity, oxygen shortage, salt stress, hormone and gene-regulated physiological changes, thus further inducing a series of physiological and biochemical reactions in cells, and finally manifesting a change for the status of plant development and metabolism [22]. Therefore, the knowledge of the principles for plant signal transduction can be enriched and proved directly through determining the changes of cellular Ca²⁺ concentrations and understanding the spatial-temporal rules for such changes after cells' stimulus reception. Meanwhile, such researches can also provide some certain theoretical instructions to regulate plant development and metabolism.

In this experiment, it can be seen that membrane Ca²⁺ orderly distributed, and Ca²⁺ concentrations decreased dramatically in vacuoles and intercellular gaps 30 min after PEG stress. Therefore, it can be observed that the cytosolic Ca²⁺ partially originated from cells' outside and partially came from vacuoles (cellular Ca²⁺ sinks). With PEG stress continued, the cytosolic Ca²⁺ concentrations also elevated increasingly, and the cells transformed. 75 min after PEG stress, membrane breakage occurred in the sections where Ca²⁺ concentrated in large quantities. Numerous reports indicated that at the early stage of cold-stress, Ca²⁺ concentrations increased significantly in cytoplasm, but the concentrations of Ca²⁺ precipitates decreased in vacuoles and intercellular gaps. Therefore, it was speculated that under the stimulus of cold stress, Ca²⁺ in vacuoles and intercellular gaps can access into cytoplasm through Ca²⁺ transport channels in the membranes and other pathways as a second messenger, initiating and controlling relevant gene transcriptions and regulations aiming at cold stress resistance, thus leading to a series of physiological and biochemical reactions such as the occurrence of cold-tolerance relevant proteins, the enhancement of some enzymes' (including catalase, ATPase and so on) activities, and finally improving plant capacities of cold-resistance [50,65–73]. It can be speculated in this experiment that Ca²⁺ presents similar mechanism in plant drought and cold resistances, because the changes of Ca²⁺ distribution were identical both at the early stage of drought stress and under cold stress. It was also reported that Ca²⁺ applies such mechanism to enhance plant drought resistance as following: on one hand, Ca²⁺ can strengthen the stability of plant cellular structure through its direct effects; on the other hand, Ca²⁺ can also affect cellular protective systems, damage their functions and maintain the metabolic balance of such bio-radicals as active oxygen through its indirect effects [52]. Such report was also supported by the changes of Ca²⁺ distribution concluded in this experiment. The cytosolic Ca²⁺ concentrations and distributions can play active roles in regulating outside environments if they change in optimal extents. While if such changes overpass certain extents, they'll bring destructions and disturbs (such as the changes of cytoskeleton and membrane structure) to normal cellular structures and functions, altering membrane permeability and finally leading to the imbalance of cellular metabolism and the occurrence of disorders [39]. It was also found that the direct addition of Ca²⁺ carriers (such as A23187) or other elements to cell plant lines can lead to the elevation of cellular Ca²⁺ concentrations or the destruction of Ca²⁺ stable imbalance both inside and outside cells, thus further inducing cell death, demonstrating that there exist certain relationships between Ca²⁺ and cell death. In this experiment, with the advancement of drought stress, cytosolic Ca²⁺ concentrations continuously enhanced, and cells continuously transformed and finally disintegrated, which indicated that at the early stage of drought stress, the drought-resistance of cells enhanced with the elevation of cytosolic Ca²⁺ concentrations, while the increasing enhancement of Ca²⁺ concentrations may serve as an induction element for cell death during drought stress [67–73].

It was found in wheat seedling that under normal conditions, the chloroplast presented elliptical shapes with orderly-laminated granules and small amounts of Ca²⁺ distributions. With the advancement of drought stress, Ca²⁺ concentrations in chloroplasts increased, and chloroplasts transformed and finally disintegrated [52]. In another report, Zhong [51] also found the changes of chloroplast super-microstructures while observing the cellular super-microstructures in drought-stressed sugar-cane leave cells, which further clarified Wang's results. As important places for photosynthesis, chloroplasts can unavoidably produce such active oxygen as superoxide radicals during the process of photosynthesis. Under normal physiological conditions, the production and elimination of these radicals are balanced, bringing no damage to plants. While under drought stress, such balance can be destroyed, thus leading to the peroxidation of membrane lipids, causing membrane leakage and the destruction of cellular super-microstructures. In this experiment, the Ca²⁺ concentrations in chloroplasts increased because of chloroplasts' effects on cytosolic Ca²⁺ concentration regulation as Ca²⁺ pumps, and the increased Ca²⁺ concentrations in chloroplasts are also beneficial for the stabilization of their membranes. However, with the advancement of drought stress, chloroplasts' structures were destroyed, especially that the transformation of their granular laminas may make great contributions to cell death. The membrane structures in chloroplast granular laminas play pivotal roles in luminous energy absorption, transmission and conversion. So, the damage of such structures may lead to metabolic disorders and finally result in cell death [27–35,57–62].

At the early stage of drought stress, despite of the elevation of Ca²⁺ concentrations in nucleus, the structures of nucleus remained stable, which demonstrated the roles nucleus played in cytosolic Ca²⁺ concentration regulation. However, with the continuous elevation of Ca²⁺ concentration, chromatins conglomerated and nucleus membranes disintegrated. Through conducting the research of the effects of Ca²⁺ on the reconstruction of non-cellular nucleus systems, Zhao and Zhang [52] found that the addition of EGTA to Ca²⁺-contented samples led to the elimination of chromatin conglomeration in striping sperms, while without such EGTA addition, the chromatins were highly-conglomerated in Ca²⁺-contented striping sperms. Therefore, it can be speculated that high Ca²⁺ concentrations may play inhibited roles in the elimination of chromatin conglomeration. Such speculation can be expressly proved through observing the TEM slices produced in this experiment. Zhao's experiment results were also proved by Wang's reports (Wang and Zhang [50]) regarding the researches of the relationships between Ca²⁺ and the changes of nucleus super-microstructures in wheat seedling leaves under drought stress, which is similar to the results observed in this experiment.

It was showed that although various stresses can all lead to the increase of cytosolic Ca²⁺ concentrations, they have different origins for Ca²⁺ concentration increase. In the heat-shock reactions of tobacco, the increase of cytosolic Ca²⁺ concentrations originated from two Ca²⁺ sinks inside and outside the cells, respectively. While in the process of low temperature-induced alfalfa cold acclimatization, such increase originated from Ca²⁺ sinks outside the cells, and Ca²⁺ can be released from cellular Ca²⁺ sinks even through touch and wind. In this experiment, after the implementation of EGTA, Verapamil and TFP pretreatments, although ABA could induce the elevation of cytosolic Ca²⁺ concentrations, the extents for such elevation were significantly lower than such extents under only-ABA induction without pretreatments. Therefore, it can be directly concluded that ABA-induced elevation of cytosolic Ca²⁺ concentrations originated mainly from Ca²⁺ sinks outside sells and partially from cellular Ca²⁺ sinks. Ca²⁺ signals can be created directly from Ca²⁺ sinks outside cells and released from cellular Ca²⁺ sinks, or it can also be jointly produced by both types of Ca²⁺ sinks. Currently, pharmacological experiments are mainly conducted for the investigation of the origin of Ca²⁺ concentration elevation due to technological limitations. It has been clarified that the changes of cytosolic Ca²⁺ concentrations mainly resulted from Ca²⁺ released from cellular Ca²⁺ sinks in such aspects of experiments as pollen tube growth [24], oxygen shortage [19], cold-shock [30] and so on. While as to the researches regarding plant defensive reactions, almost all the results manifested that Ca²⁺ outside cells played paramount roles as showed in the experiments applying such plant materials as soybean, carrot, Petroselinum sativum and tobacco. In these experiments, Ca²⁺ concentrations lowered, leading to the partial inhibition of phytoalexin synthesis [56]. However, the elevation of Ca²⁺ concentrations induced by cold stimulation, drought stress, oxygen shortage and salt stress mainly originated from the joint effects of extracellular Ca²⁺ influx and release of Ca²⁺ from cellular Ca²⁺ sinks [41–46]. Through TEM, it can also be observed in this experiment that the elevation of cytosolic Ca²⁺ concentrations could be induced by 50 μM ABA treatments to seedling leaf cells, while if extracellular Ca²⁺ influx was blocked by other reagents, the elevation of cytosolic Ca²⁺ concentrations could also be inhibited to some extents, showing that ABA-induced cytosolic Ca²⁺ concentrations elevation mainly originated from extracellular Ca²⁺. Contrary to the results of TEM observation, under digital con-focus microscopic observation, ABA-induced Ca²⁺ concentrations were not affected by any element 30 min after 100 μM Verapamil pretreatments. The following two possibilities could lead to such results: (1) different Ca²⁺ channels in the membranes have different sensitivities to Verapamil; or (2) ABA-induced Ca²⁺ concentrations elevation could originated from the release of Ca²⁺ from cellular Ca²⁺ sinks, which can be demonstrated by a 2 mM EGTA pretreatment experiment in maize cuticles. The results of such experiment showed that ABA-induced Ca²⁺ concentrations elevation could be inhibited by EGTA, indicating that such elevation might mainly originate from extracellular Ca²⁺ influx [1–5,31–36].

As a small protein molecule with a molecular weight of 17 kDa, CaM is universally-existed in plants. In this experiment, ABA-induced cytosolic Ca²⁺ concentrations elevation was inhibited by TFP, which represents an inhibitor to CaM and its analogues, showing that CaM participated in ABA signal transduction. Then, how could TFP inhibit ABA-induced cytosolic Ca²⁺ concentrations elevation through antagonizing CaM? As widely existing in plant cells as well as cell walls, cellular CaM serves as the most important Ca²⁺ receptor protein for initiating downstream Ca²⁺ signal events. In this experiment, through applying TFP, only the downstream Ca²⁺ signal events could be affected, while the changes of Ca²⁺ concentrations could not be blocked. Therefore, the effects of TFP on the pathway of ABA signal transduction could only be implemented through antagonizing CaM in cell walls. It was also proved in the experiment applying broad bean cuticle cells that Ca²⁺ concentrations could be elevated by extracellular CaM, and therefore CaM was considered to have the functions of signal molecules as extracellular signal elements, which could cause the changes of Ca²⁺ concentrations [66–73]. In this experiment, it was also showed that ABA could induce and transmit the signals of Ca²⁺ concentrations elevation through extracellular CaM.The total report here clearly provided a direct evidence for calcium ion as an important signal. However, the related molecular mechanism for ABA's correlation with extracellular CaM in inducing the changes of Ca²⁺ concentrations remains to be further studied and clarified at the next step.