Effects of Cadmium Stress on Key Enzymes Involved in Nitrogen Metabolism and Nitrogen, Phosphorus,Potassium Accumulation of Different Varieties of Rice

Wei HUANG,Jianwei PENG*,Rong GONG,Haibo TUO，Yanmi FAN

1.College of Resources and Environment,Hunan Agricultural University,Changsha 410128,China;

2.Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources Use,Changsha 410128,China;

3.Hunan Provincial Key Laboratory of Plant Nutrition in Common University,Changsha 410128,China;

4.National Engineering Laboratory on Soil and Fertilizer Resources Efficient Utilization,Changsha 410128,China

Effects of Cadmium Stress on Key Enzymes Involved in Nitrogen Metabolism and Nitrogen, Phosphorus,Potassium Accumulation of Different Varieties of Rice

Wei HUANG1,2,3,4,Jianwei PENG1,2,3,4*,Rong GONG1,2,3,4,Haibo TUO1,2,3,4，Yanmi FAN1,2,3,4

1.College of Resources and Environment,Hunan Agricultural University,Changsha 410128,China;

2.Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources Use,Changsha 410128,China;

3.Hunan Provincial Key Laboratory of Plant Nutrition in Common University,Changsha 410128,China;

4.National Engineering Laboratory on Soil and Fertilizer Resources Efficient Utilization,Changsha 410128,China

In this study,the pot experiment was used to explore the differences of activity of key enzymes involved in N metabolism and NPK accumulation under Cd stress during the tilliering stage of differen varieties of rice.The results showed that: Cd stress could increase the NPK concentration of different rice type in the tillering stage,while Shen-Liangyou 5867,Yongyou 5550 and Wu-Yunjing 27 showed the highest amplification respectively.Morever,Cd stress can also contribute to the activity of NR,GS,GOGAT increasing.A s for NR,the Cd stress significantly contribute to NR activity increasing of Huang-Huazhan and Yongyou 538 but is not significant for Wu-Yunjing 27,Shen-Liangyou 5867 and Yongyou 5550,however,the difference among them is not obvious.However,for the activity of GS,Cd stress promote the GS activity.Huang-Huazhan and Wu-Yunjing 27 with low activity in Cd normal level are the most sensitive.Meanwhile the difference between two treatment is the most significant.To the contrary,restrain the GS activity of Shen-Liangyou 5867,Yongyou 5550 and the difference is not significant.And under Cd stress,either difference reached significant in GS activity.Cd stress also improve the activity of GOGAT, Wu-Yunjing 27 showed the highest inprovement which showed the lowest GOGAT activity under Cd normal level.Cd stress on rice growth and development of adverse,make its lower seed setting rate,1 000 grain weight decreased,resulting in different degrees of reduction of output of rice.

Rice;Cadmium stresses;Tillering stage;Nitrogen metabolism

C admium(Cd)is one of the most toxic heavy metals, which polluted soil,compare with other elements,Cd can not be degraded by microorganism,but can be transferred and transformed in the environment with stronger ability of migration from soil to plants and strong activity in the environment.Meanwhile, Cadmium(Cd)is a heavy metal element harmful to animals and plants with high mobility and low poisoning concentration,and is potentially toxic to all biological organisms[2].Rice has strong capability of Cd absorption[8]. When it grows in the soil of toxic heavy metal pollution,excessive toxic heavy metals accumulate in its root,stem, leaf and grain,which have serious impacts on rice growth to decrease rice quality.Rice is the staple food of more than 65%of the population in China, and rice suffered from Cd pollution greatly in China in recent years,which not only harmed the health of the people[6],but also had taken a heavy toll on regional economic of rice production.Therefore,it is of great signifi-cance to study on rice Cd pollution[1]. The background value of Cd in the nature is low[3],but with the development of the national industrialization,unreasonable emissions of the"three wastes",bad treatment of solid waste, wastewater irrigation,agricultural usage of sludge,fertilizer containing heavy metals and so on,more and more paddy fields are polluted by Cd in China with a tendency to increase[7], promoting large releasing of Cd into farmland ecosystem,causing sharp increasing of Cd content in farmland soil to excessive level.According to reports,there were 113×104hm2of cultivated land polluted by Cd,covering 11 provinces and cities[4],and about 100 billion tons of grain were reduced by heavy metals every year,and 120 billion tons of grain were polluted causing at least 20 billion yuan of economic losses[5].Because the rice has relatively high Cd tolerance and the characteristics of easy accumulation of Cd, especially when the rice plants did not show the Cd poisoning and the production is not affected,the excessive accumulation of Cd in rice is not easy to found,and how to effectively control the Cd content in rice is the key to the sustainable development of human society[9-12].In this study,different varieties of rice as experiment material, through pot experiment,the changes of nitrogen metabolism and related enzyme activity under the condition of Cd stress were investigated,to discover the effects of nitrogen metabolism enzyme on rice Cd tolerance and the physiological reasons of rice Cd tolerance.

Materials and Methods

Experiment design

Experiment was conducted in teaching base Yunyuan ofHunan Agricultural University,and the testing soil was red yellow earth developed from the quaternary red clay,which contained 18.669 g/kg of organic matter, 1.561 g/kg of total nitrogen,183.247 mg/kg of available nitrogen,0.634 g/kg of total phosphorus,20.443 mg/kg of rapid available phosphorus,49 g/kg of total potassium and 88 mg/kg of rapid available potassium,with the PH of 4.73.The testing rice had five varieties,which were Huang-Huazhan, Wu-Yunjing 27,Shen-Liangyou 5867, Yongyou 5550 and Yongyou 538.The soaking of seeds and accelerating germination were operated on 27 May, transplant on 12 June and harvest on 7 October.

Using pot experiment,5 levels of 2 factors were set,including 10 treatments,and the two factors were Cd pollution and Cd of normal levels,and 5 levels were the 5 testing rice varieties.The experiment was operated in glass network room of teaching and practicing base of agricultural resources department of Hunan Agricultural University.In pot experiment, 20 cm×30 cm porcelain culture pots were selected,within 6.25 kg of dry soil sieved with the 1 cm sieve.Before filling with soil,CdCl2solution of 1 mg Cd was added into every 1 kg of soil in Cd pollution treatment,and no Cd was added in Cd normal level treatment, and the NPK fertilizer was applied as base fertilizer for one time,and the application amount of NPK fertilizer were 0.15,0.1 and 0.15 g/kg,respectively.There were 15 repeats,including 150 pots,in randomized block arrangement.There were 3 hills in every pot,and 3 plants every hill,managed by normal method.The sampling was conducted in active tillering stage to determine index,the seeds were investigated to calculate yield of every pot after harvest,being dried in the sun and threshed.

Determination item and methods

Basic soilT otal nitrogen,total phosphorus,total potassium,available nitrogen,rapid available phosphorus, rapid available potassium,organic matter and pH value were determined.Total nitrogenPhosphorus and potassium of plant:they were digested and heated with H2SO4-H2O2,and total nitrogen was determined by semi-micro kjeldahl determination;total phosphorus was determined by vanadium molybdate yellow colorimetric method; total potassium was determined by flame photometry.

Key enzyme activity of Nitrogen metabolism determinationNitrate reductase(NR):nitro salicylic acid colorimetry was applied.NR activity of unit fresh weight=X×V1/(V2×W×t) [μg/(g·h)].In the formula,Xrefers to the total nitrite nitrogen of reaction liquid enzyme(ug),andV1refers to buffer liquid volume of enzyme extraction (ml);V2refers to enzyme fluid volume of enzyme reaction;Wrefers to fresh weight of samples;trefers to reaction time(h).

Glutamine synthetase(GS):the standard curve of γ-glutamyl hydroxamate(Sigma)was drawn to determine GS,and enzyme activity represent with micromole number of γ-glutamyl hydroxamate.xrefers to total quantity of γ-glutamyl hydroxamate generated by reaction liquid enzyme,the rest was the same as that above.

Glutamate synthase(GOGAT): GOGAT was determined by the method of Singhet al[11].Enzyme activity represent with micromole number of NADH of catalyzed oxidation every hour and every gram(mol,h-1g-1FW);the quantity of NADH(μg)consumed every 30 s in ultraviolet spectrophotometer to consume as a unit of enzyme activity.The formula=X×V1/(V2×W×t)[μg/(g·30 s)].Xrefers to NADH(μg)consumed in 30s after enzymatic reaction,V1refers to the buffer fluid volume(ml);V2refers to crude enzyme fluid volume of enzyme reaction (ml);Wrefers to fresh weight of sample;trefers to reaction time of 30 s.

Seeds investigation and yield calculation.Spike number,seed numbers,thousand seed weight and setting percentage of each pot were investigated.

Data analysis

All test data were settled and the drawing was conducted by Microsoft Excel 2003,and differences among different treatments were analyzed by one-way analysis of variance of SPSS 10.0 software.Significance testing was operated byLSDmethod(P＜0.05),all the results were expressed with average values.

Results and Analyses

Effects of Cd stresses on dry matter accumulation of rice in tillering stage

Tillering stage refers to the period of plant from emergence to jointing, mainly the growth of vegetative organs as root,leaf,tillering and so on,and is the period of the most vigorous and largely developmental root growth and vegetative organs starting to grow.Toleaves,all the leaves near roots growed in this period,accounting for 3/4 of total leaves.Therefore,Analysis on the accumulation of NPK is crucial to plant growth trend research and grow element.

As Table 1 showed,Cd stress affected rice the absorbing mineral elements.As a whole,adding Cd treatment could promote rice growth of tillering stage,increased amount of dry matter accumulation of aerial part was shown.However,its influence degree depended on the type of different genotypes and elements.Except Huang-Huazhan and Wu-Yunjing 27, nitrogen accumulation amounts of aerial part of other three varieties were improved by Cd stress,thereinto,the difference of two Shen-Liangyou 5867 treatments was significant,and the differences of the other four varieties were not significant.Meanwhile,Cd stress decreased P accumulation of aerial part,and the accumulation degree varied with varieties,the dry mater of Huang-Huazhan,Wu-Yunjing 27 and Shen-Liangyou 5867 decreased,but ascending range of Yongyou 5550 to 33%,effects of Cd stress on the potassium accumulation of aerial part varied with genetic types, K dry matter accumulation of Huang-Huazhan and Wu-Yunjing 27 was decreased by Cd stress,while the K accumulation of other varieties was increased.The range of increasing and decreasing varied with genotype,and only standard curve of K accumulation of Wu-Yunjing 27 treatments showed significant difference.

As Table 1 showed,dry matter accumulation of one treatment showed differences with different genotypes. Nitrogen dry matter of Huang-Huazhan and Wu-Yunjing 27,which were in normal Cd treatment,were 0.675 7 kg/pot and 0.582 8 kg/pot,without significant difference.Accumulation of Yongyou 5550 was the lowest, difference of Shen-Liangyou 5867 and Huang-Huazhan were extremely significant different,but differences among the other 3 varieties were not significant.In Cd stress treatment,N of Wu-Yunjing 27,Huang-Huazhan and Shen-Liangyou 5867 were not extremely significantly different.In normal Cd treatment,P dry mater accumulation of Shen-Liangyou 5867 was the highest,and that of Yongyou 5550 was the lowest,without significant difference.Except Huang-Huazhan and Yongyou 5550,the other three varieties showed extremely significant differences.In addition,in normal Cd treatment,P dry mater accumulation of Yongyou 5550 was the highest,and that of Shen-Liangyou 5867 was the lowest,and they were significantly different.Huang-Huazhan,Wu-Yunjing 27 and Yongyou 538 did not show extremely significant difference.K accumulation of Yongyou 5550 in normal Cd treatment was the highest,and that of Shen-Liangyou 5867 in Cd stress treatment was the highest,and the other varieties did not show extremely significant differences.

Effects of Cd stress on key enzymes of nitrogen metabolism of rice in tillering stage

Effects of Cd stress on NR of rice leaf in tillering stageNitrate reductase(NR)is a vital oxidoreductase associated with nitrogen metabolism, which could reduce nitrate ions into nitrite ions.

As Fig.1 showed,Cd stress affected the tillering stage of rice leaf nitrate reductase activity,the affecting extent of which varied with varieties,thereinto,Cd stress promoted the activity im-provement of Huang-Huazhan,Wu-Yunjing 27 and Yongyou 538;it had inhibiting effects on Wu-Yunjing 27, Yongyou 5550 and Yongyou 538,except Yongyou 538,the treatments of the other varieties showed extremely significant differences.Different varieties of one treatment showed great differences,NR activity of Shen-Liangyou 5867 and Yongyou 5550 of normal Cd treatment were the highest without significant differences,but were extremely significantly different from the other three varieties.Cd stress treatment also showed the similar rule,the NR activity of Shen-Liangyou 5867 and Yongyou 5550 were the highest,and showed significant differences from other three varieties.It indicated that Cd stress had slight NR activity inhibition effects on rice of tillering stage,which had high NR activity,and had promotion effects on varieties of low activity in normal Cd treatment.NR activities of one treatment were determined by gene.

Table 1N,P,and K concentration of different rice type under Cd stress

Table 2Effects of Cd stress on yield and components

Effects of Cd stress on GS of rice leaf in tillering stageGlutamine synthetase(GS)is another important enzyme of nitrogen metabolism in leaves,which could catalyze ammonium ion and glutamic acid to synthesize glutamine.In the study of higher plants,GS was often defined as a necessary enzyme to ammonia assimilation of plant.

As Fig.2 showed,the sensitivities of rice leaves in tillering stage to Cd were different.Thereinto,Huang-Huazhan and Yongyou 538,which had the lowest GS activities in normal Cd treatment were the most sensitive to Cd stress.Cd stress promoted the increasing of their GS activity,and the difference of the two treatments was extremely significant.However,It had inhibition effects on Shen-Liangyou 5867 and Yongyou 5550,GS activity of which were relatively high in normal Cd treatment,and the differences of Yongyou 5550 in the two treatments were not significant.Therefore,the effects of Cd stress on different rice varied with genotype.

Effects of Cd stress on GOGAT of rice leaf in tillering stageGOGAT is an enzyme could reduce α-ketoglutaric acid into glutamic acid using glutamine as ammonia donor,which is the main way of plants producing glutamic acid[12-13].

As Fig.3 showed,Cd stress promoted the improving of GOGAT activity of rice in tillering stage.The improving range of Wu-Yunjing 27 with the lowest GOGAT activity in normal Cd treatment,and GOGAT activity of the other four varieties improved slightly.

Seeds investigation and yield calculation

As Table 2 showed,the plant height,spike length,seed setting rate, thousand seed weight and average output per pot of rice in normal Cd treatment were greater than that of Cd stress treatment.It indicated excessive Cd was harmful to the growth and development of rice,decreased the setting percentage and thousand seed weight,which lead to certain reduction of output of rice.

Conclusions

In this study,on the whole,Cd stress can improve NPK content in rice of the tillering stage,and the increasing range of Shen-Liangyou 5867, Yongyou 5550 and Wu-Yunjing 27 were the highest.Meanwhile,Cd stress can increase activity of NR,GS and GOGAT.To NR activity,NR activity of Huang-Huazhan and Yongyou 538 was improved by Cd stress,the effects of which were not obvious, however,the differences among each other were not significant.To GS activity,Huang-Huazhan and Wu-Yunjing 27,GS activity of which were relatively low in normal treatment were the most sensitive to Cd stress.Cd stress could improve their GS activity,and the differences of two treatments were extremely significant.It had inhibition effects on Shen-Liangyou 5867 and Yongyou 5550,GS activity of whichwere relatively high in normal Cd treatment,and the differences of each other were not significant.Any variety under Cd stress,difference of GS did not reach significant level,GOGAT activity of rice in tillering stage was improved under Cd stress,thereinto,increasing range of Wuyunjin 27,of which the GOGAT activity were the lowest in normal Cd treatment,was the largest.And Cd stress was harmful to growth of rice,and could decrease the setting percentage and thousand seed weight,which leads to reduction of output of rice,and it consistent with previous research results[14-17].In conclusion,promoting effects of Cd stress on nitrogen accumulation had the same law with the effects on three enzyme activity,which indicated that Cd stress affected nitrogen metabolism through affecting activity of nitrogen metabolism related enzyme as NR, GS and GOGAT.Therefore,researches on changes of nitrogen metabolism and its related enzyme were of great significance to investigate effects nitrogen metabolism enzyme on rice Cd tolerance and its physiological reasons.

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Responsible editor:Yong XU

Responsible proofreader:Xiaoyan WU

鎘脅迫對不同水稻品種氮代謝關(guān)鍵酶活性和植株氮磷鉀積累的影響

黃維1，2，3，4，彭建偉1，2，3，4*，龔蓉1，2，3，4，庹海波1，2，3，4，范艷咪1，2，3，4
（1.湖南農(nóng)業(yè)大學資源環(huán)境學院，湖南長沙410128；2.農(nóng)田污染控制與農(nóng)業(yè)資源利用湖南省重點實驗室，湖南長沙410128；3.植物營養(yǎng)湖南省普通高等學校重點實驗室，湖南長沙410128；4.土壤肥料資源高效利用國家工，湖南長沙410128）

該研究以不同品種的中稻為研究對象，通過盆栽試驗，探討Cd脅迫對不同水稻品種分蘗期幾種氮代謝關(guān)鍵酶活性和植株氮磷鉀積累量的差異性。結(jié)果表明：鎘脅迫能夠提高分蘗期水稻體內(nèi)NPK含量，分別以深兩優(yōu)5867，甬優(yōu)5550和武運粳27的提高幅度最高。同時鎘脅迫一定程度上也促進了NR，GS，GOGAT活性的提高。就NR活性而言，鎘脅迫明顯地促進了以黃華占和甬優(yōu)538NR活性的提高，對武運粳27，深兩優(yōu)5867，甬優(yōu)5550影響不明顯，但相互間的差異都沒達到顯著水平。而對于GS活性，其中以鎘正常處理下GS活性較低的黃華占和武運粳27對鎘脅迫最為敏感，鎘脅迫促進了他們GS活性的增加，且兩處理間差異達到極顯著。但對鎘正常處理下GS活性較高的深兩優(yōu)5867和甬優(yōu)5550表現(xiàn)出抑制作用，但是相互間差異不明顯。而且鎘脅迫下的任一品種，相互間GS差異并未達到顯著水平。

水稻；鎘脅迫；分蘗期；氮代謝

"十二五"國家科技支撐計劃項目（南方丘陵山地農(nóng)區(qū)農(nóng)業(yè)面源污染防控技術(shù)集成與示范）(2012BAD15B04)；湖南省高校創(chuàng)新平臺開放基金項目（水稻對生長關(guān)鍵期高溫逆境的氮代謝響應(yīng)機理研究）(13K061)；湖南省自然科學基金項目（水稻對生長季極端高溫氮代謝的響應(yīng)機理研究）(12JJ6016)。

黃維（1989-），男，碩士研究生，從事施肥原理與技術(shù)研究，E-mail：273232375@qq. com。*通訊作者，彭建偉（1970-），男，博士，教授，主要從事植物營養(yǎng)生理與環(huán)境生態(tài)研究，E-mail：314967900@qq.com。

2015-04-15

修回日期 2015-06-20

Supported by"Twelfth five-year"National Science and Technology Support Project (2012BAD15B04);Open Fund Project of Innovation Platform of Hunan Province Colleges and Universities(13K061);Natural Science Fund Project of Hunan Province (12JJ6016).

*Corresponding author.E-mail:314967900@qq.com

Received:April 15,2015 Accepted:June 2,2015