Bionsors and Bioelectronics 24(2009)1537–1542
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Bionsors and
Bioelectronics
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i o
s
A non-invasive and rapid ed vigor bionsor bad on quantitative measurement of superoxide generated by aleurone cell in intact eds
Xuejun Liu,Caiji Gao,Da Xing ∗
MOE Key Laboratory of Lar Life Science &Institute of Lar Life Science,South China Normal University,Guangzhou 510631,China
a r t i c l e i n f o Article history:
Received 8April 2008
Received in revid form 25June 2008Accepted 26June 2008
Available online 4July 2008Keywords:Bionsor
Chemiluminescence Rapidity Seed vigor Superoxide
a b s t r a c t
温暖人心的话
Superoxide generated during the early imbibition is an excellent marker for evaluating ed vigor.In this paper,a new principle bionsor for non-invasive detection of ed vigor bad on quantitative mea-surement of superoxide via lective probe 2-methyl-6-(p -methoxyphenyl)-3,7-dihydroimidazo [1,2␣]pyrazin-3-one (MCLA)-mediated chemiluminescence (CL)was developed.The bionsor,which ud a compact single-photon counting module (SPCM)to collect the CL signal,could evaluate ed vigor in vivo .Benefiting from the high CL efficiency of MCLA reacting with superoxide and high nsitivity of the SPCM technique,the trace superoxide generated by dry eds under storage state can be detected to achieve rapid and non-invasive determination of the ed vigor.In comparison with the traditional methods for fast measuring ed vigor bad on measurement of physiological and biochemical properties,our pro-pod technique has significant advantages such as low cost,simplicity,convenient operation and short time consuming.To demonstrate the utility of the system,it was applied to evaluate MCLA-mediated CL of three different plant species wheat (Ze Yu No.2),maize (Tai Gu No.1and 2)and rice (Jing Dao No.21)eds with different degrees of aging.The experimental results suggested that there was an excellent pos-itive correlation between the ed vi
gor asssment from quantitative TTC-test and the detection bad on MCLA-mediated CL of superoxide measurement.The new principle of ed vigor measurement is a challenge and breakthrough to conventional method of ed vigor determination and may be a potential technique of the next generation ed vigor detection.
©2008Elvier B.V.All rights rerved.
1.Introduction
In order to feed the growing population we need to constantly improve grain unit production on the limited arable land,and high-quality ed provided an important guarantee for enhancing crop unit production.Seed vigor testing,which is the cornerstone of all other ed technologies,tells whether a crop of eds is worth col-lecting,whether handling procedures are correct,and how many potential edlings are available for regeneration.Therefore,the determination of eds vitality is very important for improving agricultural yield.
So far two types of methods are commonly ud for detec-tion of ed vigor:one is by measuring the ed germination conditions and another is through detecting the physiological and biochemical indicators of ed.The traditional methods,which are bad on the germination such as germination r
ate,edling length,edling weight and accelerated aging germina-
∗Corresponding author.Tel.:+862085210089;fax:+862085216052.E-mail address:xingda@ (D.Xing).
URL:www.lar./xingda.htm (D.Xing).tion,have veral disadvantages such as time consumption and inability to distinguish the dormancy ed.Now,most available methods for measuring ed vigor are bad on detecting physi-ological and biochemical indicators,such as electric conductivity,dehydrogena activity,ATP content and acid phosphoestera activity,the above-mentioned assays may involve a longer pro-cessing time,destruction of the tested ed,a high cost and specialized skill,and the results are easily affected by environ-mental factors especially by the containing water in ed (Peters,2000).
In order to carry out the rapid and non-invasive ed vigor deter-mination,we must investigate the biochemical reactions of ed under store state.Several studies have documented the produc-tion of reactive oxygen species (ROS)during ed storage in the dry state (Bucharov and Gantcheff,1984;Hendry,1993;McDonald,1999;Pukacka and Ratajczak,2005).It has been reported that the production of ROS during ed germination in fact repre-nts an active,beneficial biological
reaction that is connected with high germination capacity and vigorous edling develop-ment (Schopfer et al.,2001).This view is supported by the finding that a strong ri in ROS relea takes place in the healthy,actively germinating ed and adding extrinsic H 2O 2can resume
0956-5663/$–e front matter ©2008Elvier B.V.All rights rerved.doi:10.1016/j.bios.2008.06.040
1538X.Liu et al./Bionsors and Bioelectronics24(2009)1537–1542
the ed vigor(Schopfer et al.,2001;Ogawa and Iwabuchi,2001), namely ROS is related to ed vigor.The active roles of ROS dur-ing ed germination have been further demonstrated as follows: (1)H2O2promotes ed germination by the oxidated decom-position of the germination inhibitors prent in the pericarp (Ogawa and Iwabuchi,2001;Oracz et al.,2007).(2)ROS pro-duction by germinating eds reprents an active,development control physiological function,protecting the emerging edling against pathogen attack(Schopfer et al.,2001).(3)ROS can func-tion as cellular cond mesngers that are likely to modulate many different proteins,leading to a variety of respons(Mori and Schroeder,2004).However,an enzymatic dismutation step mustfirst take place to convert the free radical O2•−to the more stable H2O2derivative that is required for a viable long-range cell-to-cell signal or for passing membranes(Allan and Fluhr,1997). It is possible that there is a
n endogenous mechanism to gener-ate ROS in dry ed,and this kind of ability gradually declines with the aging of eds.In our laboratory,the previous work has propod a new ed vigor detection method bad on the mea-surement of ROS generated during the early imbibition(Chen et al.,2003).Our latest rearch has suggested that the ROS(most notably superoxide)generated by the catalyzing of NDAPH oxi-da(NOX)rver as an intrinsic nsor of NADPH,hence,the superoxide can indicate the intensity of NADPH in ed(Liu et al.,2007).It has been reported that the plasma-membrane NOX is involved in ROS generation in rice(Frahry and Schopfer,2001), maize(Andrés et al.,2007)and wheat(Agarwal et al.,2005;Hao et al.,2006;Yang et al.,2007)and the NOX coding genes in the species have been quenced.Thus,detecting the superox-ide via2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2␣] pyrazin-3-one(MCLA)-mediated chemiluminescence(CL)can be ud to determine this endospermic crops ed vigor.
The endosperm of many corps such as rice,maize,wheat and barley grain consists of two differentiated cell types.In the mature ripe grain,an outer layer of living aleurone cells envelopes the dead cells of the starchy endosperm(Oln et al.,1995;Bethke et al.,2000).The aleurone cells function both as storage tissue and for cretion of hydrolytic enzymes,which upon activation dur-ing germination help break down storage tissues(Oln,1998). ROS-mediated cell death in cereal aleuro
ne cells accelerates the relea of hydrolytic enzymes,thus promoting the ed germina-tion(Bethke and Jones,2001;Angelika et al.,2002).The ROS is a mesnger molecule for ed germination,so the measurement of superoxide generated from aleurone cells can be ud to realize this kind of ed vigor.
In this study,a novel fast and portable ed vigor bionsor with new quantitative,nsitive and low cost assay for the asssment of ed vigor was developed bad on our previously mentioned principle for measuring ed vigor using MCLA-mediated CL.Our approach utilizes preci measurement of superoxide via the n-sitive and lective MCLA-mediated CL during the early imbibition. Furthermore,the single-photon counting technology bad on photomultiplier tube(PMT)has been widely ud to achieve lumi-nescence detection(Magrisso et al.,2006;Wang et al.,2007).Bad on the above principle and technique,a rapid optical measure-ment system using89C55single-chip microcomputer as control center,is designed for ed vigor detection in this paper.It can detect CL using single-photon counting module(SPCM)with high nsitivity and low signal-to-noi ratio basing on ultra-weak luminescence detection technique.Benefitting from the SPCM technique and the high CL efficiency of MCLA reacting with super-oxide,the bionsor was high nsitive,and achieved multi-sample detection,simplified operation.It can be ud as a feasible
mea-surement system for ed vigor and may have wide application foreground.2.Materials and methods
2.1.Theory for measurement of ed vigor using MCLA-mediated
CL
In the endosperm eds,the active aleurone layer cells are located in the outermost layer.In the cells intrinsic NADPH oxi-da(NOX)(Liu et al.,2007),and its activity can be activated and amplified rapidly by the influx of Ca2+in the early imbibition.The NOX catalyzes NADPH to generate superoxide anion,which can relea outside(Sagi and Fluhr,2006;Pei et al.,2000)and can be measured by lective CL probe MCLA.
MCLA-mediated CL reflects the NADPH concentration
NADPH+O2NOX
−→NADP++H++O2•−(I) MCLA+O2•−
k1
剃胡子−→MCLA−+O2+h (CL)(II) The equation of kinetics of bisubstrate enzyme-catalyzed reaction (I)
d[O2•−]
d t
= =
内能的单位
max[NADPH][O2]
K A m[O2]+K B m[NADPH]+[NADPH][O2]
(1)
max is the maximum rate or maximum velocity;K A m is the Michaelis constant for NADPH;K B m is the Michaelis constant for O2.In the mea-surement condition the intensity of oxygen[O2]is invariable.The value of max stands for the activity of NOX,which arrives at culmi-nation after ed mature and declines gradually during the storing. On the assumption that the activity of NOX has not declined,we can consider Eq.(1)as a function that the ratio of superoxide anion pro-duction(d[O2•−]/d t)changed with NADPH concentration[NADPH], the intensity of O2•−([O2•−])fluctu
ate with[NADPH],the intensity of MCLA-mediated CL[CL]real-time reflects the change of[O2•−]. We can conclude that there exists a positive correlation between [CL]and[NADPH].If NOX activity decreas during the ed conr-vation,[O2•−]will decline.In this ca,we can also conclude that there exists a positive correlation between[CL]and[NADPH].NOX catalyzes NADPH and O2to generate superoxide anion,namely, NOX prents in eds and rves as an intrinsic NADPH nsor. Therefore,the production of superoxide anion can be acted as an indicator of the NADPH concentration in ed,or the composite effects of the change of NOX activity and NADPH concentration, which are directly related to ed vigor(ISTA,1999).
During the ed germination,the early steps in rerve mobi-lization are-oxidation and glycolysis pathway(Angelika et al., 2002),NADPH will produce in the process.It had been reported that the aleurone layer of cereal grains stores significant amounts of triglycerides that are mobilized shortly after the grain imbibes water(Kristoffer and Allison,2003).And NADPH concentration is higher in the high vigor ed,namely NADPH is an indicator of ed vigor(Reuzeau and Cavalie,1995).So,we can investigate the super-oxide anion by the MCLA-mediated CL in ed aleurone layer cells instead of NADPH content to asss the ed vigor.
2.2.Materials and reagents
Regular rice eds(Jing Dao No.21)was obtained from Guang-dong Academy of Agricultural Sciences;maize(Tai Gu No.1/2)and wheat(Ze Yu No.2)eds were obtained from Shanxi Academy of Agricultural Sciences.The rice eds were harvested in July 2001,2002,2004,2005,and2006,respectively;the maize and wheat eds were harvested in2006.All the samples were taken in parate cloth bags,stored in a desiccator with silica gel and kept in room temperature(15–28◦C).Seeds in all experiments were lected and prepared carefully.MCLA was purchad from
X.Liu et al./Bionsors and Bioelectronics24(2009)1537–15421539
狼和兄弟
Tokyo Kai Kogyo Co.Ltd.;MCLA concentrations were bad upon ε430nm=9.6×103(mol L−1cm−1).2,3,5-Triphenyltetrazolium chlo-ride(TTC)and dimethyl sulfoxide(DMSO)were purchad form Sigma–Aldrich,China(Shanghai,China).MCLA was dissolved in DMSO and stored in−20◦C.
2.3.Sample preparation
2.3.1.Artificial accelerated aging
High temperature,high humidity accelerated aging is a good predictor of grain life and quality,and ac
celerated aging procedures using high temperatures(38–45◦C)and100%relative humidity (RH)in maize and wheat eds are adequate for predicting ed viability(McDonough et al.,2004).According to the above methods the maize and wheat eds were kept in perforated plastic boxes. The boxes were put in a growth chamber(model E7/2;Conviron, winnipeg,MB,Canada)with100%RH at41◦C for different time from0to96h to get different degrees of accelerated aging eds.
2.3.2.Quantitative TTC reduction test
The pericarp was parated from the endosperm.The endosperm including aleurone layers were parated from dry crop(rice,maize)grains as described previously(Li et al.,2007). 1mL endosperm cell suspension(106cells mL−1),1mL0.8%(w/v) TTC and4mL0.5M Na2HPO4–KH2PO4(pH7.4)were put into a 15mL centrifuge tube with a screw cap.The tubes were mixed by using a vortex mixer for30s and incubated at37◦C for2h, 2mL acetoacetate was then added.The tubes were shaken using the vortex mixer and then centrifuged at2150rpm(975×g)for 20min.The supernatant was transferred to a test tube.One more extraction of the retained endosperm cells with2mL acetoacetate was done as above(more extraction did not show any red color). Finally,the optical density(OD)of the supernatant was measured at 485nm using a spectrophotometer(Lambda35,UV/VIS spectrom-eter,Perkinelmer,America).The control sample was ud to zero the spectrophotometer.The triphenyl
formazan(TF)concentration was then obtained from the standard curve.
2.4.Measurement of MCLA-mediated CL
MCLA-mediated CL was measured in rice eds with different natural aging degrees(harvested in different years),as well as maize and wheat eds with different artificial aging degrees(kept in high temperature and high humidity for different time).Before the measurement of CL,the eds of equal number were weighed, put in a quartz cuvette and kept in sample ponds of the dark box, the appropriate amount of MCLA solution(thefinal concentration is1M)was injected equably into the cuvette.After incubation for 10min in darkness the measurement began.The whole data acqui-sition time of each experiment was about5–10min.The intensity of CL was normalized to cps/g dry weight(cps/g dw).All operations were performed at37◦C and in darkness.The results of measure-ments prented in the text were the average CL intensity offive replicates.
2.5.Bionsor system
2.5.1.Concept of operation
Two different operation modes(remote and local control)were optional.In the remote control,the oper西游记的故事
ation of the main instru-ment was carried out through a PC;while in the local mode,the operation was accomplished on the front panel of the instrument. Atfirst,parameters including MCLA concentration,the position of the sample,the experimental duration and temperature were t. The intact eds and MCLA mixture solution were in the sample pools;the measurement was bad on ultra-weak luminescence detection device(PMT)and the single-photon counting technol-ogy and the results were showed on the front panel(local control mode)and the PC display(remote control mode)in terms of MCLA-mediated CL.There were16sample poolsfixed on the sample tray droved by rotary stepper motor in accordance with the require-ments of changing the sample.So,the efficiency was achieved though measuring veral samples in one time and avoiding the repeated operation during the sample switching.The measurement process of CL signal was divided into two steps:background survey and meterage of mix signal including auto-oxidation CL and back-ground.CL irradiation dynamics curve was obtained by subtracting background from mix signal.In the bionsor,since the CL signal was stable during veral hours,the CL intensity was chon dur-ing the early measuring time as required.Finally,the ed vigor was determined by comparison with the relative average CL intensity.
2.5.2.System hardware design
The bionsor measured the ed vigor by detecting the lumi-nescence produced by the reaction of superoxide generated during the early imbibition with the CL probe MCLA.The major hardware system block diagram of the bionsor was shown in Fig.1.The sys-tem was mainly compod of the following hardware parts:stepper motor driving,temperature control,sample position identification and change system,single-photon technology modules,data acqui-sition and processing system,sample ponds and mask castte et al.According to the emission wavelength of CL probe,SPCM (PMT,MP-962,Perkinelmer,Wiesbaden,Germany)was adopted to receive the CL signal,who typical value of dark counting was 20–30cps,detection wavelength range165–850nm,which includ-ing the MCLA emission peak(463nm).In order to achieve real-time multi-sample detection,the rotary stepper motor was employed to switch the sample ponds.Proportional-integral-derivative(PID) technology was applied to control the system temperature and the precision reached0.1◦C.Data acquisition and processing was accomplished by micro control unit(MCU)(AT89c55)in the local control mode and PC in the remote control mode.
2.5.
3.Design of stepping motor control system
小时了了
In our system,the driving circuit of stepper motors included univoltage drive circuit,high and low voltage switching drive cir-cuit,constant current chopped drive circuit and so on.Among them the constant current chopped drive circuit was best perfor-mance,in this approach the driving current waveform was greatly improved,so that the basic constant current output,lower system power consumption and high power efficiency were realized(You et al.,2005).The system employed a single-chip sinusoidal subdivi-sion two-pha stepper motor driver application specific integrated circuit(ASIC)(TA8435H;Toshiba,Tokyo,Japan),which could be droved by two-pha stepper motor,with the circuit simple and reliable.The chip also had the following features:wide voltage range(10–40V),the average output current up to1.5A,the peak current up to2.5A,employed the pul-width modulation(PWM) chopper drive mode,with clockwi direction and counterclock-wi rotation control functions,as it can be done1/8subdivision operation,the vibration and noi generated by two-pha hybrid stepping motor in low-speed operation were overcome.The step-per motor drive control diagram was shown in Fig.2.Single chip micyocomputer(MCU)controlled the stepper motor driving,mean-while,adjusted the position of sample pool accurately by infrared signal the launch and testing devices installed beside the tray,the position calibration was executed each rotary lap.The ensured that SPCM was always in the same sample pool position during measurement,avoiding the error induced by the position warp.
1540X.Liu et al./Bionsors and Bioelectronics 24(2009)
1537–1542
Fig.1.MCLA-mediated CL-bad ed vigor detection bionsor block diagram illustrating major hardware subsystems.Power supplies omitted for sake of clarity.
2.5.4.Dark sample chamber
The structure of dark sample chamber was described in the top left corner of Fig.1.The dark chamber included periphery light tight dark box and center chamber,which were a rubber cylinder and an adiabatic plastic tray stick together,driven by the rotary stepper motor.There were 16mi-oval cylinder grooves on the verge of the cylinder.The silver–gilt grooves wall functioned as a viewfinder to collect the light to the lens.The mi-oval cylinder grooves were ud to place the sample measuremental cuvette.There was a temperature nsor and temperature adjusting system at the center of the chamber.
2.5.5.Temperature control
The difference between the environmental temperature and the t drove the executant (thermal energy converter (TEC))to work to stabilize the environmental temperature at the t temperature.TEC can stabilize the temperature through refrigerating one side and heating the other side by changing current direction during the process of working.Proportion integral control was utiliz
ed
to
Fig.2.Schematic of stepper motor drive system.
reduce static state error and improve control precision.In the sys-tem,the temperature control range is 15–40◦C and precision can reach 0.1◦C.
3.Results and discussion
3.1.Respon of CL intensity to temperature
The relationship between sample temperature and the intensity of CL was first investigated.As shown in Fig.3,average CL intensity incread with the temperature and reached the maximum at 37◦C then declined.So,this kind of ed vigor determination had a rela-tionship with temperature,in order to achieve maximum efficiency,the propod measuring temperature was 37◦C.3.2.Correlations between CL and ed vigor
According to the principle of ed vigor asssment via TTC-test,TTC can be reduced by reductive hydrogen (NADPH and
NADH)
Fig.3.Effect of the temperature on the MCLA-mediated CL.Concentration of O 2,138–166mmHg.Data are the means ±S.E.of five replicates.
X.Liu et al./Bionsors and Bioelectronics 24(2009)1537–15421541
generated in ed,to form TF,a red fluorescent compound.The TF concentration was measured by light intensity at the wave-length of 485nm,and the value of OD 485was ud to stand for the concentration of reductive hydrogen,which positively corre-lated with ed germination.We measured the concentration of reductive hydrogen of rice eds harvested in different years via TTC-test,also examined MCLA-mediated CL induced by superox-ide generated from aleurone cells in the eds.Interestingly,there existed a same trend between MCLA-mediated CL and TF concen-tration (Fig.4A).Correlative analysis indicated that this kind of MCLA-mediated CL had an obviously positive correlation to TF con-centration (stand for ed vigor quantitatively)(Fig.4B).So,our methods using MCLA-mediated CL can be ud for rapid and non-invasive detection of ed vigor.3.3.Accelerated aging test
To further demonstrate the ability of the developed bionsor,contrast experiments for measuring ed vigor using the bion-sor and quantitative TTC-test were performed for three different plant species maize (Tai Gu No.1and 2)and wheat (Ze Yu No.2)eds under the temperature of 37◦C and O 2concentration of 138–166mmHg.The ed vigor from the bionsor exhibited good accordance with that from the quantitative TTC-test for different plant species (Table 1and Supplementary figure
往生人1).In addition,the statistical results showed that the measurement time using the bionsor was less than 10min,while a visible ed vigor value via TTC-test required more than 2h.Furthermore,the developed bionsor with remote control had the predominance of low cost,simple and convenient operation,as well as original and
com-
Fig.4.Correlation between average MCLA-mediated CL intensity and quantitative TTC-test (shown as TF concentration)of rice eds harvested in different years.Experimental conditions:temperature,37◦C;concentration of O 2,138–166mmHg.Data are the means ±S.E.of five replicates.
Table 1
Linear analysis of relationship between MCLA-mediated CL intensity and TF concentration Plant species R S.D.N P
Rice
Jing Dao No.210.9931953.993195 6.73518E −4Wheat
Ze Yu No.20.9824167.35842550.00279Maize
Tai Gu No.10.9848563.46639550.00223Tai Gu No.2
0.98701
55.6285
5
寻找时传祥0.00177
R :correlation coefficient.P :value-probability (that R is zero).N :number of data points.S.D.:standard deviation of the fit.
pact appearance.The merits would make the new bionsor have powerful competition in rapid examination and non-invasive inspection of ed vigor development for precision agriculture.The new principle of ed vigor measurement is a challenge and break-through to conventional method of ed vigor detection bad on monitoring physiological and biochemical properties,and it may be a potential technique of new generation ed vigor measurement.4.Conclusions
In this study,a novel ed vigor bionsor bad on quantita-tive measurement of superoxide in vivo was developed.The novel features of the bionsor described here include:(1)the bion-sor was bad on a new principle-measuring ed vigor using CL from the reaction of MCLA with superoxide generated in eds,the detection results from this method could be less interfered by the environmental conditions (temperature and RH during storage and the water content of the ed)when compared with conventional method for measuring ed vigor.Becau of the high CL ef
ficiency of MCLA reacting with superoxide and high nsitivity of the SPCM detection technique,the new method let us detect the trace super-oxide in dry intact ed under storage state.The procedure of our new method is just putting the eds into the MCLA solution and after incubation for veral minutes in darkness then collecting the chemiluminescence,and it only needed 10–20min to accomplish the measurement.But the conventional method of TTC-test needs a certain time imbibition (usually over 2h)to activate the ed and to accumulate the red reduzate TF for naked-eye obrvation,or further using a complicated extraction progress to gather the TF for quantitative analysis via light density determination under spec-trophotometer.So,in comparison with the conventional method,the operation of our propod method for ed vigor determination is less time-consuming,much easier and convenient.The notable advantages of the bionsor are rapid and nondestructive which are significant for the ed vigor determination of large number of ed species and rare species.(2)The only reaction reagent in the pro-pod method is MCLA,theoretically the cost of each test is less than 0.5cent,which is very low.The new method has the merits of low cost,simple and convenient operation,and remote control,which would make it have the powerful competition in fast,non-invasive inspection of ed vigor and development and wide application in precision agriculture.(3)The bionsor is portable becau of the u of MCU technique.(4)The bionsor has an important appli-cation.The main grain crops such as rice,wheat a
nd maize belong to endosperm eds and have NOX,which is capable of catalyzing superoxide generation.So,the categories of ed vigor can be measured using the new bionsor.In the future,this method can be further extended to other types of NOX containing eds.(5)The bionsor accomplished wonderful vigor measurement bad on the comparison between CL intensity and corresponding TF analy-
