Effects Of Exogenous Ca～(2+) On In Vitro Pollen Germination Under Salt Stress

Sexual reproduction of higher plant is a complex biology process which is regulated both spatially and temporally. Pollen germination on the stigma, tube elongation in the style and fertilization in the ovary are important stages of the process. The whole process is probably the most important event that ensures reproduction of flowering plants. Increasing evidence proved many varieties of metal ions could affect pollen germination and tube growth such as calcium ions (Ca²⁺). It is well known that calcium ion is essential in the growth medium of pollen tubes, permitting elongation in a range of concentration. Besides, calcium plays as an important signal substance to regulate pollen germination. Calcium has been considered not only as one of the indispensable macronutrients to regulate growth and development, but also as a second messenger in the coupling of many extracellular signals and intracellular biochemical and physiological events. Salt stress disrupts ionic and osmotic homeostasis of plant cells. The disorder of homeostasis occurs at both cellular and whole plant levels which lead to molecular damage, growth inhibition and even death. For plants, one of the most important strategies to survive in salt environment is to re-establish the ionic and osmotic homeostasis. However, it is not completely clear whether salt stress affects reproduction of plants. If salt stress inhibits any state of pollination, plants will not fruit and the yield will be reduced.In the present study, pollens were incubated in liquid medium and were used to study effects of exogenous Ca²⁺ on pollen germination under NaCl stress. The main results of this study are as follows:1. Effects of NaCl and adding Ca²⁺ treatments on pollen germination and tube growthLilium pollen germination and tube growth were significantly inhibited under 25 mmol/L NaCl treatment. However, 0.1 mmol/L NaCl could markedly suppress pollen germination and tube growth of Arabidopsis thaliana. The results show that NaCl treatment inhibited pollen germination and tube growth at a low concentration, compared with seeds and plants.NaCl-induced decrease in pollen germination was overcome by addition of calcium. Consequently, the reduction in pollen germination and tube growth rate under NaCl stress was less in presence of calcium. It was suggested that NaCl stress inhibited the vital role of Ca²⁺ on pollen germination. While Ca²⁺ concentration was in the range of 6～20 mmol/L, the inhibition increased with the increase of concentration.On the contrary, there was a significant difference when plant grew at high NaCl concentration. It suggested that pollen was more sensitive to NaCl and the inhibition effect of NaCl to pollen was mainly ion toxicity rather than osmotic stress.2. Effects of NaCl and adding Ca²⁺ treatments on Ca²⁺, Na⁺ and K⁺ contents of pollenNaCl markedly decreased the Ca²⁺, K⁺ contents and increased Na⁺ content, leading to decreasing the K⁺/Na⁺ ratio. NaCl stress usually disrupts the ionic homeostasis of Na⁺, Ca²⁺ and K⁺ in the cell. NaCl enhanced the Na⁺ absorption and suppressed Ca²⁺ and K⁺ playing the key role in pollen germination. The NaCl-induced decrease in Ca²⁺ content could inhibit the regulation of Ca²⁺ in pollen germination, such as keeping the stability of membrane and K⁺ uptake.While addition of Ca²⁺ significantly alleviated the inhibition of NaCl, that is Ca²⁺ increased the pollen concentration of K⁺ and decreased the uptake of Na⁺ to ensure successful germination.3. Effects of NaCl and adding Ca²⁺ treatments on permeability of pollen plasmalemma and MDA content of pollenMembrane permeability reflects the degree of membrane damage of plants. NaCl stress induced increase in the membrane permeability of pollen and MDA content, suggesting that membrane integrality and stability was damaged under NaCl stress. Subsequently, this led to the increase of Na⁺ absorption through the plasma membrane. Besides, pollens were stained using Evan's blue. The results showed that pollens under NaCl stress were stained deeper than the control pollens suggesting membrane damage ocurred.Favorable effects of Ca²⁺ application was apparent on membrane permeability. Addition of Ca²⁺ markedly reduced the membrane permeability and this was beneficial to keep trans-membrane transportation.4. Effects of NaCl and adding Ca²⁺ treatments on SOD,amylase activity and proline content of pollenNaCl induced the decrease of activities of SOD. This indicates that the scavenging capacity of free radical and reactive oxygen species in pollens was lower than control, and this led to the damage of the cells. While exogenous Ca²⁺ markedly increased SOD activity in pollen under salt stress and was benefit to increase the salt tolerance.The NaCl-induced decrease of amylase activity led to inhibiting starch hydrolysis, and pollen would be lack of substances as well as energy. However, calcium application increased starch hydrolysis in pollen compared with NaCl treatment and offered pollens hydrolysate and energy that were required for pollen germination and tube growth.Proline content in pollens was decreased with NaCl treatment compared with control. That indicated that NaCl stress inhibited proline metabolism. On the contrary, proline transferred into other substances to promote pollen germination in control pollen. However, the positive influence of calcium addition was significantly enhanced proline metabolism. Therefore, proline content of control was significantly lower than that of NaCl treatment.5. Effects of spraying NaCl and adding Ca²⁺ treatments on pollen germination on stigma and seed setting rateSpraying NaCl to flower, the total pollen on the stigma was lower than control treated with spraying distilled water. Besides, this led to a decrease in the seed setting rate. While addition of Ca²⁺ significantly increased the total pollen on the stigma and the seed setting rate.In conclusion, significant difference was observed in the responses of pollen germination, tube elongation to NaCl and adding calcium treatments. This shows that the effects of adding calcium treatment on pollens were different from those of NaCl treatment. Investigations of ion content and enzyme activities indicate that NaCl treatment induced a less ability for pollen germination. All results indicated that proper Ca²⁺ concentration could be selected to enhance the salt tolerance abilities of pollen by not only maintaining the Ca²⁺ homeostasis and a high K⁺/Na⁺ ratio in pollen, but also declining the damage of pollen membrane permeability.