Perception of salt stress in plant cells induces a change in the free cytosolic Ca2+, [Ca2+]cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H+ concentration, [H+]cyt, are closely linked to the [Ca2+]cyt dynamics under various stress signals. In this study, salt‐induced changes in [Ca2+]cyt, and [H+]cyt and vacuolar [H+] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca2+] and [H+] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium‐binding benzofuran and acetoxy methyl ester of 2′, 7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6‐carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca2+]cyt in the salt‐tolerant cv. Pokkali than in the salt‐sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca2+]cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca2+]cyt measurements in rice also suggest that Na+ should be sensed inside the cytosol, before any increase in [Ca2+]cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca2+]cyt and that osmotic stress sharply decreases [Ca2+]cyt in rice. The [pH]cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H+‐ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H+‐ATPase in these two rice cultivars.

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

Arbuscular mycorrhiza (AM) is known to be a mutually beneficial plant-fungal symbiosis; however, the effect of mycorrhization is heavily dependent on multiple biotic and abiotic factors. Therefore, for the proper employment of such plant-fungal symbiotic systems in agriculture, a detailed understanding of the molecular basis of the plant developmental response to mycorrhization is needed. The aim of this work was to uncover the physiological and metabolic alterations in pea (Pisum sativum L.) leaves associated with mycorrhization at key plant developmental stages. Plants of pea cv. Finale were grown in constant environmental conditions under phosphate deficiency. The plants were analyzed at six distinct time points, which corresponded to certain developmental stages of the pea: I: 7 days post inoculation (DPI) when the second leaf is fully unfolded with one pair of leaflets and a simple tendril; II: 21 DPI at first leaf with two pairs of leaflets and a complex tendril; III: 32 DPI when the floral bud is enclosed; IV: 42 DPI at the first open flower; V: 56 DPI when the pod is filled with green seeds; and VI: 90–110 DPI at the dry harvest stage. Inoculation with Rhizophagus irregularis had no effect on the fresh or dry shoot weight, the leaf photochemical activity, accumulation of chlorophyll a, b or carotenoids. However, at stage III (corresponding to the most active phase of mycorrhiza development), the number of internodes between cotyledons and the youngest completely developed leaf was lower in the inoculated plants than in those without inoculation. Moreover, inoculation extended the vegetation period of the host plants, and resulted in increase of the average dry weight per seed at stage VI. The leaf metabolome, as analyzed with GC-MS, included about three hundred distinct metabolites and showed a strong correlation with plant age, and, to a lesser extent, was influenced by mycorrhization. Metabolic shifts influenced the levels of sugars, amino acids and other intermediates of nitrogen and phosphorus metabolism. The use of unsupervised dimension reduction methods showed that (i) at stage II, the metabolite spectra of inoculated plants were similar to those of the control, and (ii) at stages IV and V, the leaf metabolic profiles of inoculated plants shifted towards the profiles of the control plants at earlier developmental stages. At stage IV the inoculated plants exhibited a higher level of metabolism of nitrogen, organic acids, and lipophilic compounds in comparison to control plants. Thus, mycorrhization led to the retardation of plant development, which was also associated with higher seed biomass accumulation in plants with an extended vegetation period. The symbiotic crosstalk between host plant and AM fungi leads to alterations in several biochemical pathways the details of which need to be elucidated in further studies.

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