Study on Effects of Calcium on Photosynthetic Characteristics and Its Subcellular Distribution in Rice at Seeding Stage

Guoqi ZHU,Yuzhu ZHANG*,Yueping ZHANG,Xiang ZENG,Xinghuai ZHANG

1.Hunan Rice Research Institute,Changsha 410125,China;2.Anxiang County Farm Bureau,Changde 415600,China

Calcium is one of a large number of mineral nutrients neces sary for plants,and it plays an important role in plant growth and development and a variety of physiological activities[1].Calcium deficiency leads to cell wall disruption,affecting cell division.Calcium in biofilms can be used a bridge between the phosphate radical of phospholipids and carboxyl of proteins,maintaining the stability of membrane structure.Calcium is also an activator for ATP enzyme and phospholipase in membranes[2].Calcium is essential for the growth of rice,and each producing 6.2-8.0 t of rice requires to absorb 24.3-35.4 kg of cal cium from the soil[3].Especially in the southern red soil,brick red soil and other calcium-deficient areas,calcium fertilizer can significantly increase rice yield and improve milled rice rate and head rice rate[4-5].The researches on physiological responses of rice to calcium and subcellular distribution of calcium will help to understand the effect mechanism of calcium on rice growth,and also contribute to further rational application of calcium in production.

There have been many reports on effects of calcium on salt tolerance,heavy metal tolerance and cold resistance of rice.However,most of them focused on the physiological regulation function of calcium under stresses[6-9].The distribution of calcium in subcellular structures of plants tissues has been a hot topic,and there have been also many related researches.Wanget al.[10]found that calcium is mainly located in the vacuoles of wheat cells,and it is also abundantly distributed in intercellular spaces.Chenet al.[11]and Mouet al.[12]studied the subcellular distribution of calcium in apples and tomatoes,and similar results were obtained.Currently,there have been rare reports on the subcellular distribution of calcium in rice cultivars with different calcium contents in brown rice,let alone on genotypic differences in subcellular distribution of calcium among different cultivars.To this end,using the rice cultivars with different calcium contents in brown rice as tested materials,the effects of calcium on photosynthetic characteristics of rice at seedling stage,as well as differences in physiological response of different rice cultivars to calcium concentration,were investigated.Moreover,the contents of calcium in organelles of rice roots and leaves were determined so as to reveal the mechanism and differences of calcium accumulation in rice from the perspective of calcium distribution in different organelles.

Materials and Methods

Materials

The rice cultivars with different calcium contents in brown rice were used as tested materials[12],including two high calcium rice cultivars(Nanyou 4 and Zhou 903)and two low calcium rice cultivars(5755-3 Yunda and 4077-2-3).They were all provided by the germplasm bank ofHunan Province.

Test design

The test was carried out in the net chamber of Hunan Rice Research Institute in 2009.The physiological responses of rice to different-concentration calcium treatments and subcellular distribution of calcium were studied with hydroculture method.The seeds were evenly sowed in germination boxes(50 seeds per germination box).After the seedlings emerged,the standard liquid nutrient was sprayed.At the three-leaf stage,the young seedlings with uniform growth were selected and transplanted to plastic buckets in diameter of 20 cm and height of 30 cm,and there were four plants in each bucket.The formula of standard liquid nutrient was the same as described by the International Rice Research Institute (Table 1).The liquid nutrient was replaced once a week.All the plants were treated with different-concentration CaCl2 (0.0,0.5,1.0,5.0 and 10.0 mmol/L)20 d after the transplanting.In the CaCl2-deficient treatment groups,the plants were treated by same-concentration (calculated as Cl-)NaCl.There were five replicates in each treatment group.

After certain days(20 d),the roots of rice plants were soaked in Na2-EDTA(20 mmol/L)for 3 h to remove adhered ions on their surface.The roots and leaves were sampled separately.The collected samples were rinsed with distilled water and then preserved at-70℃for use[13].

Determined indexes and methods

Determination ofphotosynthetic parametersThe photosynthetic parameters of second(from bottom)fully expanded leaves,with synchronous growth,were determined 15 d after the calcium treatment.The portable photosynthesis system(LI-6400,LI-COR),with red and blue light sources,was used.The determination was carried out at 9:00-11:30 under following conditions:CO2concentration of(380±5)μmol/mol,airflow velocity of 500 μmol/s and light intensity of 800 μmol/(m2·s).The determination was repeated three times.The determined photosynthetic parameters included net photosynthetic rate(Pn),stomatal conductance (Gs),intracellular CO2concentration(Ci),etc.

Subcellular distribution of calcium in different organelles of riceThe subcellular fractions in various organelles were separated using fractional classification.A certain amount of each sample was mixed with a certain amount of Tris-HCl buffer(10 mmol/L,pH 7.4).For root samples,the solid to liquid ratio was 1:4;and for leaf samples,the solid to liquid ratio was 1:2.The sample was homogenized in liquid nitrogen,and then,the subcellular fractions were separated(Fig.1).Before each centrifugation,the sample was all supplemented to the same volume with buffer.The obtained precipitates (F1,F2,F3,F4)and supernatant(F5)were used for calcium content analysis(Fig.1).

Heating digestion and analysis of residues and precipitatesThe F1precipitate (cell wall and unbroken residues)was rinsed with deionized water and then filtered through quantitative filter paper.The newly-obtained precipitate was dried at 60℃.And then,it was soaked in HNO3-HClO4(5 ml)overnight and digested at 200℃till the solution turned clarified.The solution was diluted to 50 ml with deionized water,and the calcium content in the filtrate was measured by atomic adsorption spectrophotometer.The other precipitates(F2-F5)were transferred to small beakers with deionized water and evaporated to dryness on a hot plate.After adding certain amounts of concentrated nitric acid(2 ml for each),the precipitates were digested till the solutions turned clarified.The solutions were diluted to 10 ml with deionized water respectively.Finally,the calcium contents were determined.

Table 1 Formula of standard liquid nutrient

Data analysis

The general statistics were performed using Excel 2010;the variance analysis was performed using DPS 7.05;the multiple comparisons were performed using LSD method.

Results and Analysis

Effect of calcium on photosynthesis of rice

Calcium deficiency(CK)has significant negative effects on photosynthesis in rice,such as low net photosynthetic rate.The effects of calcium concentration on net photosynthetic rate were different among different types of rice cultivars (Fig.2).In high calcium cultivar,the leaf net photosyntheticrate wasincreased with the increased calcium concentration,but there was certain difference between the two high calcium cultivars.In the concentration range of 0.5-5.0 mmol/L,the leaf net photosynthetic rate was still relatively high in Nanyou 4,but its increase was relatively slow.The leaf net photosynthetic rate in Zhou 903 was increased rapidly with the increase in calcium concentration.The two low calcium rice cultivars were all sensitive to calcium concentration.When the calcium concentration exceeded 0.5 mmol/L,the leaf net photosynthetic rates in the two low calcium cultivars were all decreased with the increased calcium concentration.It was indicated that the calcium demands and tolerances of high calcium cultivars were significantly higher than those of low calcium cultivars.The effects of calcium on leaf net photosynthetic rate were similar to those on stomatal conductance.When the calcium was deficient,the stomatal conductances of Zhou 903 and 5755-3 Yunda were lowest.Increased calcium concentration improved the stomatal conductance of Zhou 903,a high calcium rice cultivar.However,the further increased calcium concentration reduced the stomatal conductances.The effects of calcium on intercellular CO2concentration were opposite to those on leaf net photosynthetic rate.Compared with the control,the intercellular CO2concentration was first reduced and then increased with the increased calcium concentration.For the low calcium cultivars,the inflection points all appeared at the calcium concentration of 0.5 mmol/L;but the inflection points of the two high calcium cultivars appeared at 1.0 (Nanyou 4)and 5.0 (Zhou 903)mmol/L,respectively.Among the four rice cultivars,the intercellular CO2concentrations in high calcium treatment group(10.0 mmol/L)were all higher than those in low calcium treatment group(0 mmol/L).It suggested that high-concentration calcium ions promoted the aggregation of intercellular CO2,thus the activity of RuBP carboxylase was inhibited and the utilization efficiency of CO2was reduced,thereby leading to excessive retention of CO2in intercellular spaces.

Leaf transpiration rate was trended to be increased first and then decreased with the increase in calcium concentration.The transpiration rate of Zhou 903 was highest at the calcium concentration of 1.0 mmol/L.When the calcium was deficient,the transpiration rate was lower;when the calcium concentration was increased to 0.5-1.0 mmol/L,the transpiration rate was increased sharply;but when the calcium concentration exceeded 10.0 mmol/L,the transpiration rate was sharply decreased to the level as calcium was deficient.For the two low calcium cultivars,the transpiration rates were highest at the calcium concentration of 0.5 mmol/L;and then,their transpiration rtes were decreased gradually with the increase in calcium concentration.

Effect of calcium on subcellular distribution of calcium in rice

Calcium contents in subcellular organelles of rice roots and leaves of different-concentration calcium treatment groupsAs shown in Table 2,the calcium contents in various organelles of roots were all increased significantly with the increase in calcium concentration.Under the same calcium treatment concentration,the calcium contents in various organelles all ranked as cell wall(F1)＞ribosome and intracellular soluble components (F5)＞plastid (F2)＞ mitochondria(F4)＞nucleus(F3).Compared with the CK,low calcium treatment(0.5 mmol/L)significantly increased calcium content in cell.The cellular total calcium content was increased by 29.06%.Among the organelles,the in-creasesofcalcium contentswere largest in cell wall,ribosome and intracellularsoluble components,while were smallest in plastid,nucleus and mitochondria.In the treatment group with calcium concentration of1.0 mmol/L,the cellular total calcium content was increased by 76.92%.Among all the organelles,the increases of calcium contents were all relatively large,and in plastid,nucleus and mitochondria,the increases of calcium contents are even higher than 50%.In the treatment groups with calcium concentrations of 1.0 and 5.0 mmol/L,the variations of calcium contents in various organelles were basically the same,indicating higher calcium tolerance of roots.The high calcium treatment (10.0 mmol/L)significantly increased the cellular total calcium content in rice roots,and the cellular total calcium content reached 6.11 mg/g FW.Particularly,the calcium content in cell wall was increased greatly,and the calcium content in cell wall in the 10.0 mmol/L treatment group was five time higher that in the 0.5 mmol/L treatment group.It is fully demonstrated that under high calcium stress,the protective effect of cell wall,as the outermost layer of cell,is most obvious.The calcium contents in ribosome,intracellular soluble components,plastid,nucleus and mitochondria were also increased in varying degrees.

Table 2 Calcium contents in organelles of rice roots of different-concentration calcium treatments mg/g FW

Table 3 showed that the variations of calcium contents in various organelles of rice leaves were similar to those of rice roots.However,the cellular calcium content in leaves was higher than that in roots in overall.With the increase in calcium concentration,the leaf cellular calcium content was increased significantly.In the 10.0 mmol/L treatment group,the cellular total calcium content reached 16.26 mg/g FW,which was 2.81 times higher than that in CK.The calcium content in cellwallaccounted for75.70%-81.79%of the cellular total calcium content,so cell wall is the main storage place of calcium,which was most obvious under the high calcium(10.0 mmol/L)stress.The subcellular distribution of calcium among organelles was basically the same between rice roots and leaves.The calcium contents in various organelles were all increased gradually with the increase in calcium concentration.The subcellular calcium content in the 10.0 mmol/L treatment group was increased by 168.76%compared with that in the 0.5 mmol/L treatment group.Therefore,it was speculated that under high calcium stress (10.0 mmol/L),leaf cells were damaged to some extent,and the regulation function of cells was weakened,resulting in the accumulation of a large amount of subcellular calcium ions.

Distribution of calcium among different subcellular organelles in different-concentration calcium treatment groupsAs shown in Fig.3,when the calcium concentration was less than 5 mmol/L,the percentages of calcium contents in cell wall and ribosome and intracellular soluble components of roots were decreased with the increased calcium concentration,but the percentages of calcium contents in plastid,nucleus and mitochondria were trended to be increased.In low calcium treatment(0.5 mmol/L),the percentages of calcium contents in cell wall(F1)and ribosome and intracellular soluble components(F5)were 45.96%and 28.09%,respectively.In 10.0 mmol/L treatment group,the percentages of calcium contents in various organelles,except that in cell wall(which was increased sharply to 58.22%),were all lower than those in 1.0 mmol/L treatment group.

Fig.4 showed that the percentage of calcium content in cell wall of rice leaves exceeded 75%,and the percentage of calcium content in ribosome and intracellular soluble components was also higher.In the control(CK)and low calcium (0.5 mmol/L)treatment groups,the cellular total calcium contents were all lower,and the calcium contents in cell walls all accounted for more than 80%of the cellular total calcium contents.In the 1.0 and 5.0 mmol/L treatment groups,since the calcium ions were combined with chloroplasts and other organelles,the relative percentages of calcium contents in cell walls were reduced.When the calcium concentration was increased to 10.0 mmol/L,the calcium content in cell wall was sharply increased again,so the percentage was also increased.

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Differences in subcellular distribution of calcium among different rice cultivars

Differences in subcellular distribution of calcium among roots of different rice cultivarsWith the increase in calcium concentration,the cellular total calcium contents in the two types of rice cultivars were all gradually increased(Table 4).However,both the cellular total calcium contents and calcium contents in various organelles of the two high calcium cultivars were higher than those of the two low calcium cultivars,and the differences were enlarged with the increased calcium concentration.There were also significant differences in subcellular distribution of calcium between the two types of rice cultivars.In the low calcium(0.5 mmol/L)treatment group,the calcium contents in root cell walls of the tow high calcium cultivars were higher than those of the two low calcium cultivars by 1.37 times.However,the percentages of calcium contents in root cell walls of the two high calcium cultivars were lowerthan those of the two low calcium cultivars,indicating that not only cell wall(F1)but also other organelles have strong capacities for accumulating calcium ions.In the high calcium (10.0 mmol/L)treatment group,the cellular total calcium contents in the high calcium cultivars were 1.14 times higher than those in the low calcium cultivars;the calcium contents in various organelles of the high calcium cultivars were all higher than those of the low calcium cultivars,especially in chloroplast(F2)where the calcium content was increased by 41.94%.

Differences in subcellular distribution of calcium among leaves of different rice cultivarsIn both low(0 and 0.5 mmol/L)and high (10.0 mmol/L)calcium treatment groups,the calcium accumulation capacities of high calcium cultivars were all higher than those of low calcium cultivars(Table 5).The leaf cellular total calcium contents of the two types of rice cultivars were all increased with the increase in calcium concentration.However,the cellular total calcium contents of high calcium cultivars were higher than those of low calcium cultivars,and the differences were enlarged with the increase in calcium concentration,which were consistent with the study results about rice roots.

Table 5 also showed that under condition ofcalcium deficiency (0 mmol/L),the calcium contents in chloroplasts of the two types of rice cultivars were all relatively low(＜5.0%),and with the increase in calcium concentration,the calcium contents in chloroplasts were all increased gradually.It suggested that under condition of calcium deficiency,the demand for calcium by chloroplasts was inhibited,so increased calcium concentration promoted the absorption of calcium.In high calcium(10.0 mmol/L)treatment group,the percentages of calcium contents in chloroplasts of the two types of rice cultivars were all reduced sharply,which might be due to damaged binding sites for calcium in chloroplasts under high calcium stress.

Conclusions and Discussion

Effect of calcium on photosynthesis of rice

Zouet al.[19]proposed that calcium facilitate the operation of photosynthetic products.When rice plants were shortage of calcium,carbohydrates were mainly accumulated in assimilation organs,instead of non-assimilation organs.When calcium shortage lasted for 1-2 d,although there were no changes in appearance and moisture content of rice plants,the operational speed of carbohydrates was significantly reduced.They considered that calcium promoted operation of carbohydrates might through activating ATP enzyme.The activated ATP enzyme activated the hydrogen pump,and the activated hydrogen pump promoted sucrose’s entering sieve tubes and furthertransportation.Tang[20]found that low-concentration Ca2+significantly promoted the increase in variable fluorescence(FV),enhancing the fluorescence emission of PS II at 650 nm,and Ca2+increased the chemical activity and photochemical conversion efficiency of PS II in chloroplasts.In this study,calcium showed significant effects on photosynthetic parameters of rice leaves.The leaf net photosynthetic rate was increased gradually with the increase in Ca2+concentration,but high-concentration Ca2+(10.0 mmol/L)inhibited the leaf net photosynthetic rate,which might be because that high-concentration Ca2+inhibited the formation of chlorophyll and destroyed the structure of cell membranes.This study adopted two types of rice cultivars,between which the calcium content in brown rice had significant difference[13].The leaf net photosynthetic rates of high calcium cultivars were higher than those of low calcium cultivars,and the high calcium cultivars showed higher calcium accumulation and tolerance capacities.This will provide a scientific basis for the actual application and promotion of high calcium cultivars.

Effects ofdifferent-concentration calcium treatments on subcellular distribution of calcium in rice

This experiment studied the uptake and accumulation of calcium in rice at the sub-cell level.The results showed that the calcium accumulation amounts in various organelles were increased with the increase in calcium treatment concentration.The calcium content in rice roots was lower than that in rice leaves,which might be because after entering cortical cells of leaves,calcium ions were combined with proteins to form stable calmodulin complexes,regulating many calciumdependent physiological activities in leafcells.Previous studies have shown that calmodulin(CaM)is generally abundant in vegetative organs of plants[13].The leaves are the most vegetative organ during the growth and development of rice plants.This study further illustrates that calcium accumulation in leaves is an important part of calcium accumulation in plants.

Previous studies have shown that a mechanism has been formed in plants.When the calcium concentration in cytoplasm is increased,the calcium transportsystemsinplasma membrane and vacuolar membrane will be started to transport calcium ions from cells or into vacuoles,maintaining the cytoplasmic free calcium concentration at the micromolar level[14-16].This study found that in both rice roots and leaves,the calcium contents ranked as cell wall(F1)＞ribosome and intracellular soluble components(F5)＞plastid or chloroplast(F2)＞mitochondria(F3)＞nucleus(F4).Most of the calcium was distributed in cell wall,and the calcium contents in F2,F3and F4were relatively low.In both roots and leaves,cell wall was the main storage place for calcium.In rice roots,the calcium content in cell wall accounted for more than 40%of the cellular total calcium content,while in rice leaves,the calcium content in cell wall accounted for more than 75%.These were consistent with the study results of Mouet al.[1 1]about the subcellular distribution of calcium in tomato leaves and fruits.Under high calcium (10.0 mmol/L)stress,the percentage of calcium content in cell wall was highest,which fully embodied the protective function of cell wall as the outermost barrier.This study also found that,in both low and high calcium treatment groups,the percentages of calcium contents in chloroplasts of rice leaves were all lower than those in other calcium treatment groups.

Differences in subcellular distribution of calcium among different types of rice cultivars

Tanget al.[17]studied the genotypic differences in calcium uptake and accumulation among different pepper varieties.The resultsshowed that there were significant differences in calcium content in fruit among different varieties.Gaoet al.[18]compared and analyzed the absorption dynamic model and distribution and transportation of calcium among different spring wheat varieties.They considered that there were significant differences in calcium content and distribution among different spring wheat varieties atvariousgrowth periods.In this study,the calcium contents in root and leaf cells of high calcium cultivars were significantly higher than those of low calcium cultivars,and the differences were enlarged with the increase in calcium treatment concentration.

This study showed that the cellular total calcium contents in high calcium cultivars were significantly higher than those in low calcium cultivars,and there were significant differences in calcium contents of various organs and organelles between the two types of rice cultivars.This might be because that calcium,as a second messenger in cells,has activating function for many key enzymes in plants,so high calcium cultivars need more calcium(calmodulin)to regulate many calciumdependent physiological activities in leaf cells,such as photosynthesis,cell division,messaging and hormonal regulation[21].However,the specific mechanism still needs further study.

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