ORIGINAL ARTICLE
Arabidopsis BPG2:a phytochrome-regulated gene whose protein product binds to plastid ribosomal RNAs
Byung-Hoon Kim ?Przemyslaw Malec ?
Andrzej Waloszek ?Albrecht G.von Arnim
Received:17February 2012/Accepted:22March 2012óSpringer-Verlag 2012
Abstract BPG2(B rz-insensitive p ale g reen 2)is a dark-repressible and light-inducible gene that is required for the greening process in Arabidopsis .Light pulse experiments suggested that light-regulated gene expression of BPG2is mediated by phytochrome.The T-DNA insertion mutant bpg2-2exhibited a reduced level of chlorophyll and carot-enoid pigmentation in the plastids.Measurements of time resolved chlorophyll ?uorescence and of ?uorescence emission at 77K indicated defective photosystem II and altered photosystem I functions in bpg2mutants.Kinetic analysis of chlorophyll ?uorescence induction suggested that the reduction of the primary acceptor (Q A )is impaired in bpg2.The observed alterations resulted in reduced pho-tosynthetic ef?ciency as measured by the electron transfer rate.BPG2protein is localized in the plastid stroma frac-tion.Co-immunoprecipitation of a formaldehyde cross-linked RNA–protein complex indicated that BPG2protein binds with speci?city to chloroplast 16S and 23S ribosomal RNAs.The direct physical interaction with the plastid rRNAs supports an emerging model whereby BPG2pro-vides light-regulated ribosomal RNA processing functions,which are rate limiting for development of the plastid and its photosynthetic apparatus.
Keywords Light áChloroplasts áRNA binding árRNA processing áChlorophyll ?uorescence Abbreviations
BPG2B rz-insensitive p ale g reen 2Brz Brassinazole
CaMV Cauli?ower mosaic virus ETR Electron transfer rate LHC Light-harvesting complex PSI Photosystem I PSII Photosystem II YFP Yellow ?uorescence protein
Introduction
Since plants use light as their energy source,the photo-synthetic ef?ciency is critical to their ?tness and survival.Indeed,the goal of many plant developmental processes is to obtain optimal quantity and quality of light.Those pro-cesses include leaf development,shade avoidance and chloroplast development and movement (Franklin et al.2005).In order to attain this,not only the photosynthetic gene expression,but also the expression levels of genes that are directly or indirectly related to those developmental processes are regulated by light.Based on microarray data,
B.-H.Kim (&)
Department of Natural Sciences,Albany State University,504College Drive,Albany,GA 31705,USA e-mail:bkim@https://www.doczj.com/doc/1f15966837.html,
B.-H.Kim áA.G.von Arnim
Department of Biochemistry,Cellular and Molecular Biology,University of Tennessee,Knoxville,TN 37996,USA e-mail:vonarnim@https://www.doczj.com/doc/1f15966837.html,
P.Malec áA.Waloszek
Faculty of Biochemistry,Biophysics and Biotechnology,Jagiellonian University,ul.Gronostajowa 7,30-387Krakow,Poland
e-mail:przemyslaw.malec@https://www.doczj.com/doc/1f15966837.html,.pl A.Waloszek
e-mail:andrzej.waloszek@https://www.doczj.com/doc/1f15966837.html,.pl
A.G.von Arnim
Graduate Program in Genome Science and Technology,University of Tennessee,Knoxville,TN 37996,USA
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DOI 10.1007/s00425-012-1638-6
light/dark-induced gene expression changes occur in many different cellular processes,such as nitrogen and sulfur assimilation,cell wall synthesis,protein synthesis and degradation,secondary metabolism,amino acid synthesis, hormone synthesis and responses,as well as photosynthesis (Ma et al.2001;Kim and von Arnim2006).Phytochromes are photoreceptors that play a central role in the perception of the light environment,in particular,the balance between red and far-red light(Franklin and Quail2010).
In angiosperms,assembly of the photosynthetic appa-ratus remains suppressed under dark conditions,which permits seedlings that germinate under a protective layer of soil to allocate the maximum of resources to prolonged extension growth(etiolation).Light,then,triggers a tightly orchestrated program of gene expression changes that result in the ordered assembly of the photosynthetic apparatus and thus development of the plastid organelle from a non-photosynthetic etioplast to a photosynthetic chloroplast.This program includes chlorophyll biosynthe-sis,development of thylakoid membranes and the forma-tion of thylakoid membrane stacks termed grana.All these require gene expression changes in both the plastid and the nuclear genome at the transcription and/or translation level. Analysis of chlorophyll?uorescence facilitates the assessment of plant photosynthetic activity.In particular, emission spectra at low temperatures provide information on the energy transfer to pigment-protein complexes,such as reaction centers of photosystems I and II and light-harvesting complex(LHC)II monomers(Krause1991). Conversely,kinetic analysis of chlorophyll?uorescence at room temperature is a non-invasive tool to monitor early photosynthetic events and to evaluate the physiological condition of plant organisms(Maxwell and Johnson2000).
Assembly of the photosynthetic apparatus is regulated not only by light,but also by hormones such as abscisic acid,cytokinin and brassinosteroid at least to some extent (Kusnetsov et al.1998).A previous report revealed that the BPG2gene(B rz-insensitive p ale g reen)is involved in the greening process.BPG2was identi?ed by virtue of being less sensitive to the brassinosteroid biosynthesis inhibitor, Brz(brassinazole).Mutant bpg2plants showed defects in plastid rRNA processing(Komatsu et al.2010).
We report that Arabidopsis BPG2expression is regu-lated by phytochrome.The mutation in bpg2led to a variety of changes in the?uorescence characteristics of both photosystems.The BPG2protein is localized in the plastid stroma where it mediates ribosomal RNA process-ing by interacting with rRNA.The induction of rRNA production by phytochrome during de-etiolation was described in pea and mustard plastids over40years ago during the dawn of molecular plant physiology(Scott et al. 1971;Thien and Schopfer1975).We propose that BPG2is a phytochrome-regulated gene important for assembly of the chloroplast translation apparatus and,consequently,of the photosynthetic apparatus.
Methods
Plant material and growth
Wild type,bpg2-2mutant(T-DNA insertion,SALK-068713;Alonso et al.2003)and bpg2-2mutant comple-mented by the overexpression of BPG2cDNA fused to YFP were Arabidopsis thaliana ecotype Columbia.For the experiments using young seedlings,Arabidopsis plants were grown under continuous light condition(80l mol/m2/ s)on19MS-medium containing1%sucrose and0.8% agar with or without other supplements under conditions given in the text.For the experiments using older plants, about10-day-old seedlings on Petri dishes were transferred onto soil and grown further at22°C under continuous light conditions(90l mol/m2/s).
For the induction of BPG2expression by red light pulse, 8-day-old dark-grown seedlings were illuminated for5min of red(10l mol/m2/s)or far-red(50l mol/m2/s)light. Seedlings were kept in darkness for5h and harvested for total RNA isolation.
Construction of plasmid DNA
For the BPG2-YFP fusion construct,full-length BPG2 (At3g57180)cDNA was PCR ampli?ed by Vent Poly-merase(NEB,Ipswich,MA,USA)using primers with Nco I restriction endonuclease sites(BPG2forward-Nco I, 50-acgccatggtggttttgatttcaagtacag-30;BPG2reverse-Nco I, 50-tatccatggcaacactatcagagagaa-30).The Nco I-digested PCR product was inserted into Nco I-cut of pBS-EYFP(Subra-manian et al.2006)generating a translational fusion of CaMV35S promoter-driven BPG2-YFP protein,which is named pBS-BPG2-YFP.
For the genetic complementation,pBS-BPG2-YFP was digested using Kpn I/Bam HI.The resulting expression cassette includes the promoter,BPG2-YFP coding sequence and the CaMV transcriptional terminator.This was inserted into Kpn I/Bam HI-digested binary vector pPZP222(Hajdukiewicz et al.1994)for the transformation of the bpg2mutant.
Plant stable transformation and transient expression
in onion epidermal cells
Agrobacterium tumefaciens(GV3101)harboring the appropriate binary vector was used for Arabidopsis trans-formation.Transformants were selected by growing the seedlings on MS agar medium supplemented with
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gentamicin(100l g/ml).Transient expression of BPG2-YFP in onion epidermal cells was conducted by particle bombardment as described previously(Subramanian et al. 2006).
Pigment quanti?cation
Chlorophyll and carotenoid contents were measured by following previous methods(Lichtenthaler1987).The aerial part of11-day-old seedlings(100mg fresh weight) was ground in5ml of80%acetone(20mg/ml,?nal concentration)on ice under safe green light.The solution was incubated at4°C in darkness overnight.After cen-trifugation for10min at4°C,the supernatant was diluted to1:1with cold80%acetone(10mg/ml,?nal concen-tration).Spectroscopic absorbance was measured at 663.2,646.8and470nm.The pigment contents were calculated by the following equations:chlorophyll a(C a)= 12.259A663.2-2.799A646.8;chlorophyll b(C b)= 21.509A646.8-5.109A663.2;total carotenoid=(1,0009 A470-1.829C a-85.029C b)/198.The resulting con-centrations are shown as l g/10mg plant fresh weight.Values are mean±SD from three independent experiments.
For HPLC analysis,*100mg of rosette leaves was homogenized with a pestle and mortar in5–7ml of90% acetone(v/v)with an addition of20mg of CaCO3 according to(Lichtenthaler1987).Extracts were clari?ed by centrifugation(10,000g for5min)and evaporated to dryness with gaseous nitrogen.The obtained?lm was dissolved directly in the HPLC mobile phase(solvent A). Pigments were separated with a Prostar HPLC system (Varian,Miami,FL,USA)equipped with a photodiode array spectrophotometric detector Tidas I(World Precision Instruments,Sarasota,FL,USA)and a Nucleosil100C-18 reversed phase column(4.69250mm),5l m particle size (Technokroma,Barcelona,Spain),using the solvent system described by Gilmore and Yamamoto(1991).Samples (100l l)were?ltered through a stainless steel?lter (u=0.22l m)and loaded onto the column equilibrated with the solvent A(acetonitrile:methanol:H2O72:8:1;v/v/v). The column was eluted isocratically with solvent A for 15min,followed by a one-step gradient(0–100%,2min) of the solvent B(ethyl acetate:methanol360:160,v/v)and an isocratic hold(5min)at100%B.During separation,a constant?ow rate of 1.5ml/min was ensured.The absorption spectra of the eluate(380–800nm)were recorded every0.2s.Pigments were identi?ed on the basis of both their absorption spectra and their retention times. The concentrations of carotenoid species were calculated from Beer–Lambert’s law using their speci?c extinction coef?cients at440nm(Mantoura and Llewellyn1983). Chlorophyll a contents were calculated accordingly using its speci?c extinction coef?cient at665nm(Lichtenthaler 1987).Values are mean±SE from at least three individual experiments.
Chlorophyll?uorescence and electron transport rate
The F V/F M was calculated from the ground state chloro-phyll?uorescence value(F0)and the maximum?uores-cence value(F M),where F V/F M=(F M-F0)/F M.The F M and F0were measured by using a OS-30p Chlorophyll Fluorometer(Opti-Sciences,Hudson,NH,USA).V J and S M were calculated from the numeric data of?uorescence as described in Strasser et al.(2000).
Electron transfer rate(ETR)light intensity-dependent curves were measured using a PAM210(Walz,Germany) pulse-amplitude-modulated chlorophyll?uorometer.Sour-ces of red(630–680nm)measuring(ML),adapting(AL) and saturating(SP)light were internal light emitting diodes (LED)of the apparatus.Intensities of ML and SP were below0.1and3,500l mol/m2/s of photons,respectively. After the measurement of F0and F M values,the sample was adapted for5min in the lowest AL and,in ten fol-lowing steps,for2min in increasing AL intensities.A measurement of F M has been done at the end of each adaptation period.ETR values were calculated as it is described in Schreiber(1997).
PSII ef?ciency imaging was carried out using an Open FluorCam FC800-O chlorophyll?uorescence imaging system(Photon System Instruments,Czech Rep.).For each pixel,the basic?uorescence level(F0)was measured at light?uence rate of0.1l mol/m2/s.Subsequently,the Kautsky induction kinetics of?uorescence was recorded at 200l mol/m2/s of photons.The red band of excitation light with k max=635nm was used.
Low temperature?uorescence
For77K?uorescence measurements,the Arabidopsis rosette leaves were homogenized in10mM Hepes, pH7.0and immediately frozen in capillary tubes in liquid nitrogen.Steady-state?uorescence emission spectra(600–780nm)were recorded at77K,using437nm excitation wavelength,with Perkin-Elmer LS50B spectro?uorimeter (Beacons?eld,Buckinghamshire,UK)with automatic correction for the wavelength dependence of the detection system equipped with liquid nitrogen attachment.Both excitation and emission slits were of5nm.To enhance monochromatization of the excitation beam and to elimi-nate light scattering before emission slit,the BG16and KV550optical?lters(Schott,Jena,Germany)were used, respectively.The scan rate was usually60nm/min. Three spectra were automatically averaged for a single measurement to optimize the signal/noise ratio.
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RNA isolation,Northern and RT-PCR analysis
Plant samples were harvested and immediately frozen in liquid nitrogen.Total RNA was isolated using TRIreagent (SIGMA,St.Louis,MO,USA)following the manufac-turer’s guide.For reverse transcription reactions,1l g of RQ1DNase(Promega,Madison,WI,USA)-treated total RNA and0.5l g of oligo(dT)primer were incubated at 70°C for10min and chilled on ice.To this mixture,4l l of59reaction buffer,40units of RNasin(Promega), 20l M of each dNTPs and200units of M-MLV reverse transcriptase(Promega)were added in a total volume of 20l l.The reaction was incubated at42°C for50min,and then inactivated at70°C for15min.The?rst strand cDNAs were diluted to100,and5l l(1/20of the initial amount)was used for a50l l PCR reaction.PCR was carried out under standard conditions using30cycles (BPG2)or25cycles(EF1-a)of94°C for45s,60°C for 45s and72°C for60s.20l l of PCR products was separated on a1%(w/v)agarose gel.The following primer sets were used for PCR:BPG2rt-forward,50-aagg aagttcaacctcggag-30;BPG2rt-reverse,50-aagtcgatacaggtta tactaagc-30;EF1a-forward,50-cagctaagggtgccgcc-30;EF1a-reverse,50-gtcgatcataacgaaagtctcatc-30.Control reactions with higher cycle numbers were performed to con?rm that the reactions had not reached saturation.
Northern blot analyses were carried out as described by Bollenbach et al.(2005).Five micrograms of denatured total RNA was separated on a1%agarose gel with3.3% formaldehyde and transferred to a positively charged nylon membrane.RNA on the nylon membrane was visualized with methylene blue for the veri?cation of equal loading and transfer.For the Northern hybridization of plastid16S rRNA full-length Arabidopsis rrn16DNA was labeled with32P using Rediprime II DNA Labeling System(GE Healthcare,Piscataway,NJ,USA).The probes were hybridized with immobilized RNA in the‘DIG Easy Hyb’hybridization solution(Roche Diagnostics,Mannheim, Germany).Hybridization and washing steps were carried out according to manufacturer’s instruction.The mem-brane-bound radioactive signal was detected by exposing blots to SuperRX X-ray?lm(Fuji?lm,Tokyo,Japan). Formaldehyde?xation and RNA
co-immunoprecipitation
The formaldehyde?xation and the co-immunoprecipitation methods were modi?ed from Terzi and Simpson(2009). Wild-type Arabidopsis seedlings expressing plastid targeted YFP and bpg2mutant expressing wild-type BPG2-YFP targeted to plastids were grown on MS solid media as described above.When they were10days old,about0.5g of whole seedlings were pulled out from the solid media and washed four times in cold,sterile water for1min each. Seedlings were vacuum-in?ltrated in1%formaldehyde for 15min.Formaldehyde was replaced by125mM of glycine and further vacuum-in?ltrated for5min.Seedlings were washed four times in cold,sterile water for1min each. After drying between two sheets of?lter paper,the seed-lings were either frozen in liquid nitrogen and stored in -80°C or ground in liquid nitrogen for the extraction of protein and RNA.
The samples ground in liquid nitrogen were resus-pended in1ml of RNA-coIP buffer[25mM Tris/HCl pH 8,150mM NaCl,1mM EDTA,1%Triton X-100,0.1% SDS,10l l RNasin(Promega),10l l Protease Inhibitor Cocktail(SIGMA)].The samples were centrifuged in a table top centrifuge for10min at4°C.The supernatants were centrifuged again for2min.After determining the protein concentration by BCA protein assay(Pierce, Rockford,IL,USA),10mg of crude extract with the adjusted volume of1ml was pre-cleared for3h at4°C with gentle rotation by incubating with protein A Sepharose(100l l of50%slurry)that had been previ-ously washed three times with Beads Washing Buffer (25mM Tris/HCl pH8,150mM NaCl,1mM EDTA, 1%Triton X-100,0.1mM PMSF).These pre-cleared extracts were incubated with washed protein A Sepharose (50l l of50%slurry)and5l l of anti-GFP antiserum (Molecular Probes,Eugene,OR,USA)for4h at4°C with gentle rotation.The precipitates were washed twice with Washing Buffer1(25mM Tris/HCl pH8,150mM NaCl,1mM EDTA,1%Triton X-100,0.1%SDS)for 5min each at4°C.After that,two more washing solu-tions(Washing Buffer2,25mM Tris/HCl pH8,500mM NaCl,1mM EDTA,1%Triton X-100,0.1%SDS; Washing Buffer3,25mM Tris/HCl pH8,250mM LiCl, 1mM EDTA,1%Triton X-100,1%sodium deoxycho-late)were used twice each for5min at4°C to wash out the unspeci?c binding thoroughly.Finally,the pellets were washed twice with Washing Buffer4(25mM Tris/HCl pH8,1mM EDTA)for5min at4°C.To elute the precipitates,beads were resuspended in300l l of Elution Buffer(100mM NaHCO3,1%SDS)for15min at65°C with gentle agitation.The elution was repeated with new Elution Buffer.The eluates were combined.Formaldehyde cross-link was reversed by adding5M NaCl to a?nal concentration of200mM and by incubating overnight at 65°C.The eluates were further incubated at37°C for 30min with three units of RQ1DNase(Promega).After adding protease K to a?nal concentration of50l g/ml,the eluates were further incubated at50°C for30min. Co-precipitated RNAs were puri?ed by phenol:chloro-form:isoamylalcohol extraction followed by ethanol pre-cipitation.Final pellets were solubilized in10l l of 10mM Tris/HCl(pH8).
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Radioactive labeling of cDNA probe and dot blot analysis
Ten microliters of co-precipitated RNAs were mixed with 1l l(1mg/ml)of random hexamer along with?re?y luciferase mRNA(1ng;Promega)and heated at70°C for 10min.After snap cooling on ice,the following compo-nents were added:59M-MLV reverse transcriptase buffer (5l l),10mM cold dNTP without dCTP(1l l),100l M cold dCTP(1l l),radioactive dCTP(5l l),RNasin(1l l; Promega)and M-MLV reverse transcriptase(1l l;Pro-mega).The reactions were incubated at42°C for40min. Again,1l l of M-MLV reverse transcriptase and1l l of 10mM cold dNTP(including dCTP)were added and further incubated at42°C for an additional40min.The reactions were inactivated by incubating at70°C for 15min.
Dot blots were prepared as described by Brown(2001) using the indicated concentrations of cDNA probes on a positively charged nylon membrane.Probes for EF1-a, rbcL,16S rrn and23S rrn were generated by PCR ampli-?cation of genomic DNA using primer pairs,EF1a-forward, 50-cagctaagggtgccgcc-30;EF1a-reverse,50-gtcgatcataacgaa agtctcatc-30;16Srrn-forward,50-gaaagagaggggtgccttcggg-30;16Srrn-reverse,50-acgacttcactccagtcactagcc-30;23Srrn-forward,50-gccaatgttcgagtaccaggcg-30.Partial DNA probes for atpB/E,ndhC and rpoC2were produced by enzyme digestion of Arabidopsis clones,M53H4(Eco RI/Xho I), 251N14(Sal I/Not I),and97D24(Sal I/Not I),respectively. All PCR products and restriction enzyme digested frag-ments were puri?ed and quanti?ed after agarose gel electrophoresis.Hybridization and detection were carried out with DIG Easy Hyb hybridization solution(Roche Diagnostics)under the same condition as described above for Northern analysis.
Isolation of chloroplasts and Blue Native-PAGE Arabidopsis chloroplasts were isolated from wild-type and mutant plants according to Rensink et al.(1998).Rosette leaves(10g)from3-week-old plants were used as starting material.Small amount of chloroplasts were lysed by hypotonic buffer(50mM Tris/HCl,0.01%Triton X-100, pH8.0)and the concentration of soluble proteins was determined by BCA protein assay(Pierce,Rockford,IL, USA).
Blue Native gel electrophoresis was carried out with a slight modi?cation of the protocol available from http:// https://www.doczj.com/doc/1f15966837.html,/PDF/BNPAGE.pdf.Intact chloro-plasts(equivalent of200l g of soluble proteins)were pelleted and resuspended in40l l of solubilization buffer (0.75M Aminocaproic acid,0.05M Bis–Tris,pH7.0).To solubilize membrane proteins,7.5l l of10%n-dodecyl-b-D-maltopyranoside was added and kept on ice for30min. After centrifugation(10min,4°C),supernatant was recovered and mixed with2.5l l of5%Coomassie blue in 0.5M aminocaproic acid.PMSF was also added to a?nal concentration of1mM.Samples were spun brie?y to eliminate insoluble aggregates,and20l l of supernatant was loaded onto a gradient(3.5–12%)BN-PAGE gel.Gel was run at150V in the cold room using a separate cathode buffer(50mM Tricine,15mM Bis–Tris,0.02%Coo-massie blue G,pH7.0)and an anode buffer(50mM Bis–Tris,pH7.0).To run a second dimension of SDS-PAGE,a single lane of the BN-PAGE gel was cut and incubated for 30min in10%glycerol,2%SDS,50mM Tris(pH6.8), 0.002%bromophenol blue and20mM b-mercap-toethanol.The gel strip was rinsed brie?y in the same buffer without b-mercaptoethanol,and inserted between the two glass plates before the SDS separating gel (5–20%)was cast.After the solidi?cation of separating gel and casting a stacking gel around the BN-PAGE gel strip, the second dimensional SDS-PAGE was carried out as a standard SDS-PAGE.
Statistical analyses and reproducibility
All statistical analyses(SD and SE)were carried out using Microsoft EXCEL,and treated statistically where possible as indicated above.Other non-statistical and non-quanti-tative data were performed multiple times with indepen-dent biological samples and produced similar results.
Results
bpg2mutant and gene expression
Our previous microarray study identi?ed790Arabidopsis genes that responded when seedlings are shifted from light to darkness for1–8h(Kim and von Arnim2006).Of these, 142were screened for several light/dark-related mutant phenotypes,including greening status and petiole elonga-tion under light conditions and upon shift to darkness.We found a pale green mutant with a T-DNA insertion in BPG2(B rz-insensitive p ale g reen;At3g57180;Fig.1a). The T-DNA was inserted in the?rst exon,and no full-length mRNA was detected in the mutant plants at different ages(Fig.1b).After backcrossing twice,the T-DNA co-segregated with the mutant phenotype,and this pheno-type could be complemented by constitutive expression of BPG2cDNA fused to yellow?uorescent protein(YFP; Fig.1a),con?rming that the pale green phenotype of the bpg2mutant is due to the disruption of the BPG2locus by the T-DNA insertion.The same mutant allele(Salk_ 068713)has been previously reported as bpg2-2,which
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exhibited reduced sensitivity to Brz treatment(Komatsu et al.2010).Brz,a speci?c inhibitor of brassinosteroid (BR)biosynthesis promotes chlorophyll accumulation (Nagata et al.2000).
RT-PCR analysis con?rmed our previous microarray result showing that the BPG2mRNA level was reduced upon dark treatment(Fig.1c).Prolonged dark treatment up to36h reduced the mRNA level even further,and the re-illumination of dark-adapted seedlings rapidly restored its mRNA level(Fig.1d).The light-responsive expression of BPG2is probably regulated by phytochromes,since the BPG2mRNA could be induced by a short red light pulse and reversed by far-red light pulse(Fig.1e).BPG2mRNA was found at higher level in leaves than other non-photo-synthetic organs(Fig.1f).Many light-inducible genes have multiple light-responsive cis-regulatory elements in their promoter region(Green et al.1987;Giuliano et al.1988; Mart?′nez-Garc?′a et al.2000;Luo et al.2010).The upstream region of the BPG2gene also has multiple motif elements that are known to be light-responsive in other contexts,although the classical G-box motif CACGTG is
conspicuously absent.These motifs may be responsible for the phytochrome regulation of BPG2expression(Fig.2).
BPG2protein is a soluble protein in plastids and affects the pigment composition
The BPG2-YFP fusion protein transiently expressed in onion epidermal cells co-localized with the plastid-targeted CFP protein(Fig.3a,b)supporting the chloroplast locali-zation of BPG2-GFP in transgenic Arabidopsis plants (Komatsu et al.2010).Since the BPG2-YFP signal was found in the stroma-?lled tubules,which lack thylakoids (stromules;Fig.3a inset),we conclude that the BPG2 protein is localized in the stroma fraction of plastids.We also determined the subplastid localization of transgenic BPG2-YFP protein by cell fractionation and Western blot analysis in Arabidopsis bpg2mutant plants(Fig.3c).YFP-tagged BPG2protein was detected in the soluble fraction, together with stroma proteins such as Rubisco,and was not associated with membranes(Fig.3c).The
completely
Fig.1bpg2mutant and regulation of BPG2gene expression.
a Morphological phenotype of wild type(WT),bpg2-2,and bpg2-2 complemented by BPG2cDNA fused to YFP(BPG2-YFP).
b Gene structure of BPG2and the location of T-DNA insertion in the bpg2-2 mutant.The coding sequence(black boxes)is interrupted by three introns and?anked by short untranslated regions(gray boxes).BPG2 protein contains a putative GTP-binding domain.RT-PCR could not detect BPG2transcript in the bpg2-2mutant.
c Microarray result (from Kim an
d von Arnim2006)and RT-PCR result showing repression of BPG2mRNA by darkness.Light-grown10-day-old seedlings wer
e transferred to complete darkness(D)for a given time period before harvest.L indicates the samples kept in the light. Translation elongation factor1alpha(EF1-a)served as a control.
d RT-PCR result showing th
e repressive effect o
f prolonged dark treatment(Dark)and light-inducibility after4days of dark adaptation (Light).L,the non-dark-adapted light-grown sample.e Light pulse experiment.Dark-grown8-day-old seedlings were illuminated with far-red light(FR,730nm,50l mol/m2/s,5min),red light(R, 660nm,10l mol/m2/s,5min),or red light followed by far-red light (R-FR,5min each).After light treatment,seedlings were kept in darkness for5h.As a light-inducible control dark-grown seedlings were treated with white light for5h(L).Total RNA were isolated for RT-PCR.f RT-PCR analysis usin
g total RNA samples from different organs indicates that BPG2is expressed predominantly in photosyn-thetically active organs c
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different protein banding patterns for the soluble and membrane fractions,as well as the membrane localization of marker proteins AtpB and D1,con?rmed the effec-tive separation of the soluble stroma and membrane compartments.Because transgenic BPG2-YFP comple-mented the bpg2mutant phenotype,the localization of BPG2to the stroma is most probably authentic.Despite the localization of BPG2in the chloroplast and the pale
green
Fig.2Putative light-responsive cis -regulatory
elements found in the upstream region of BPG2gene.The genomic sequence of 1kb upstream from the
transcriptional start site of BPG2was queried to PLACE (PLAnt Cis -acting regulatory DNA Elements),a database of nucleotide sequence motifs (http://www.dna.affrc.go.jp/PLACE/).Only the light-responsive elements are shown:BOXIINTPATPB (ATAGAA),GATABOX (GATA),GT1CONSENSUS (GRWAAW),IBOX
(GATAAG),IBOXCORE
(GATAA)and IBOXCORENT (GATAAGR)
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phenotype,the confocal microscopic images did not sug-gest any signi?cant difference in the number or size of chloroplasts from wild-type and bpg2mutant plants (Fig.3d).
rRNA composition in the bpg2mutant
BPG2protein harbors a putative GTP-binding domain with an unusual motif https://www.doczj.com/doc/1f15966837.html,pared to the
regular
Fig.3Subcellular localization of BPG2protein and confocal microscopic images .a Punctate localization pattern of transiently expressed BPG2-YFP in onion epidermal cells.Also shown is a bright ?eld image.b BPG2-YFP (green )and CFP fused to plastid target sequence of rbcS (red )were transiently co-expressed in onion epidermal cells.Perfect overlay is shown by yellow color (merged).c Proteins were prepared from soluble and membrane fractions of Arabidopsis chloroplasts.Coomassie staining (left panel )as well as the Western blot of atpB,psbA (D1)and BPG2-YFP (right panel )are shown.d Representative confocal microscopic images showing auto?uorescence from chloroplasts in mesophyll cells from 10-day-old seedlings of WT and bpg2mutant.Size bars indicate 30l
m
Fig.4Defect in pre-processing of rRNA and interaction between rRNA and BPG2protein.a Wild type (WT),bpg2-2mutant,and bpg2-2mutant complemented by BPG2-YFP (compl.)were used for Northern blot analysis showing the 16S rRNA band (16S)and an additional band (asterisk )that is not fully processed.Methylene blue stained nylon membrane is also shown for RNA loading Northern transfer control.b Dot blot analysis using cDNA probes after co-immunoprecipitation of RNA with BPG2-YFP or plastid-targeted YFP (cpYFP).One representative experiment from three indepen-dently replicated experiments is shown
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arrangement of G1–G5motifs,the order in BPG2is cir-cularly permutated as G4–G5–G1–G2–G3(Anand et al.2006).The GTPases of this class have been implicated in the ribosome biogenesis process and interact with ribo-somes.Two examples are Bacillus subtilis protein YqeH (Uicker et al.2007)and Escherichia coli protein YjeQ (Daigle and Brown 2004;Himeno et al.2004).Hence,BPG2might have a similar function in Arabidopsis chlo-roplasts.Indeed,consistent with a recent report (Komatsu et al.2010),we found that the mature 16S ribosomal RNA accumulates at a reduced level,and an incompletely pro-cessed form can be detected (Fig.4a).BPG2might help with pre-processing of rRNAs by directly binding to the ribosomal RNAs.To test this hypothesis,we carried out co-immunoprecipitation of a formaldehyde cross-linked complex with YFP-tagged BPG2protein in the bpg2mutant background.The co-precipitated RNAs were labeled with radioactive 32P by reverse transcription and used as probes for the dot blot hybridization to detect the species and the amount of RNAs found in the BPG2immunoprecipitate (Fig.4b).We found strong signals for 16S and 23S ribosomal RNAs in the BPG2-YFP precipi-tate.For comparison,only a low level of background was found in the control precipitate from plants with plastid-targeted YFP.The radioactive labeling ef?ciencies between the two samples were similar as indicated by similar signal intensities in the spiked-in positive control,?re?y luciferase mRNA.None of the four plastid mRNAs tested were found in the co-immunopreciptate.These data indicate that the BPG2-mediated preprocessing of rRNA may involve direct or indirect physical and sequence-spe-ci?c interaction between BPG2protein and the rRNAs.
Pigment and protein composition in bpg2mutant plastids
Photosynthetic pigments are reduced in the bpg2mutant (Komatsu et al.2010).Speci?cally,the chlorophyll a and b,and the total carotenoids content in the bpg2mutant were about 40and 60%of wild-type plants,respectively (Fig.5a).Because the chlorophyll a:b ratio was essentially normal (2.8for WT;2.7for bpg2),we conclude that there is no speci?c depletion of antenna complexes in bpg2.However,by HPLC analysis,when normalized to chloro-phyll a levels,the relative levels of the main carotenoid species were altered in bpg2.In particular,the levels of both lutein and xanthophylls which are involved in the xanthophyll cycle such as violaxanthin,antheraxanthin and zeaxanthin were elevated in bpg2.This effect was accompanied with a remarkable decrease of b -carotene accumulation suggesting that xanthophylls are formed in bpg2at the expense of b -carotene.These results may suggest that the mutants display a constitutive high-light response and/or decreased concentration of b -carotene bound in antenna complexes (Fig.5b).
Misregulation of rRNA processing in bpg2is known to reduce the abundance of certain chloroplast proteins,such as D1,LHCP and Rubisco (Komatsu et al.2010).Such a difference may directly or indirectly affect protein complex formation in the chloroplast,which we monitored by two-dimensional Blue Native-Polyacrylamide Gel Electropho-resis (BN-PAGE).A different banding pattern in the ?rst dimension of BN-PAGE was found especially in the high molecular weight complexes suggesting that the higher order protein complexes of photosystem I and II
are
Fig.5Photosynthetic pigment composition.a Chloroplast pigment content in the wild type (WT)and in the mutant bpg2-2.Chlorophyll a (Chl a),chlorophyll b (Chl b)and total carotenoids were quanti?ed.b Carotenoids in WT and bpg2from rosette leaves as quanti?ed by HPLC;n =3.Asterisk indicates statistical signi?cance by Student’s t test (p \0.05)
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constructed in a different way in the bpg2mutant(Fig.6a, b).Subsequent SDS-PAGE for the second dimension revealed roughly similar pattern of protein complexes, although subtle differences in the high molecular weight complexes could be reproducibly observed in multiple independent experiments(Fig.6b,boxes).
Alterations in photosynthetic ef?ciency in the bpg2 mutant
Reduced expression of the highly unstable photosystem II reaction center protein,D1(psbA),is a common effect of defects in the chloroplast translational apparatus(e.g., Motohashi et al.2007;Schult et al.2007)and has been observed in bpg2(Komatsu et al.2010).To examine how the mutation affected photosynthetic light harvesting and electron transport,we measured chlorophyll?uorescence parameters.Low temperature?uorescence spectra col-lected at77K provided evidence that the energy transfer to the reaction centers of photosystem II was distorted in the bpg2mutant,as indicated by the signi?cant reduction in the emission around685nm(Fig.7a,b).Pulse-amplitude-modulated(PAM)?uorescence spectroscopy in the bpg2 mutant revealed a much faster rise in chlorophyll?uores-cence than in the wild type(Fig.8a).Moreover,the basal (F0)and maximal(F M)level of?uorescence was higher and the variable level(F V=F M-F0)was lower,thus leading to a reduced F V/F M ratio(Table1).These data are indicative of a reduced quantum ef?ciency of photosystem II,as might be expected when D1is underexpressed. Additionally,the selected speci?c energy?ux parameters as estimated from chlorophyll?uorescence data were also altered suggesting that the reduction of Q A(primary acceptor of photosystem II)site is affected in bpg2.Par-ticularly,the V j parameter re?ecting the relative fraction of ‘‘closed’’PSII reaction centers,which are unable to reduce Q A,increased in the mutant by ca.25%.Simultaneously, the normalized total surface over induction curve(S M)—a measure of Q A reduction—decreased signi?cantly in bpg2
(Table1).Finally,measurement of the electron transport rate in photosystem II also demonstrated a clear reduction in the bpg2mutant,up to ca.30%of values observed in WT.Also,the light saturation point of photosynthesis was lower in the mutant,around600l mol/m2/s compared to around800l mol/m2/s in wild type(Fig.8b).Whole plant imaging con?rmed that observed alterations in chlorophyll ?uorescence between WT and bpg2(the increase of F0and F M,decrease of F V/F M)are expressed on all leaves of bpg2 mutant plants(Fig.8c).Besides the evident defect
in
Fig.6Chloroplast protein complex analysis.a The?rst dimension of a BN-PAGE using total chloroplast proteins from wild-type and bpg2 mutant plants.b Each individual lanes of BN-PAGE were cut and assembled with an SDS-PAGE gels to separate individual compo-nents of the complexes.After running,gels were stained with Coomassie Blue.The photosystem I(PSI)and the photosystem II (PSII)were localized by identi?cation of PsaD2(red arrow), LHCB2.1(green)and CP29(blue)using a tandem mass spectrometer. The boxes indicate different banding patterns in bpg2mutant c
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Fig.7Chlorophyll?uorescence in bpg2mutant plants.a77K ?uorescence spectra taken from rosette leaves of WT and bpg2-2 mutant plants.A difference spectrum is displayed,normalized at 665nm.b The data for WT and bpg2-2in panel(a)are displayed after setting the emission maximum to
1
Fig.8Photosystem II parameters.a Time resolved chlorophyll ?uorescence measured in intact dark-adapted wild type(WT)and bpg2mutant rosette leaves,normalized at maximum of emission. Inset the same data in arbitrary?uorescence units to show differences in F M values.b Electron transport rate for photosystem II in WT and bpg2mutant plants.c In situ measurements of basal(F0)and maximal (F M)?uorescence,and the ratio of variable to maximal(F V/F M) chlorophyll?uorescence in dark-adapted bpg2and WT
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photosystem II,photosystem I also appears to be compro-mised,as evident from the3nm blue-shift in the77K ?uorescence maximum around730nm(Fig.7b).Similar blue-shifts have been observed in plants defective in psaD, psaF and other PSI components(Zhang et al.1997;Hald-rup et al.2000;Sto¨ckel and Oelmu¨ller2004).In summary, the reduced ef?ciency of photosynthesis in the bpg2mutant goes a long way to explain the retardation of growth and maturation of the mutant plants.
Discussion
A previous report demonstrated that BPG2(
B rz-insensitive p ale g reen2)is an ancient nuclear-encoded chloroplast-localized protein with conserved zinc?nger and GTPase domains that supports chloroplast development and greening.BPG2is required for ef?cient processing of chloroplast ribosomal RNAs(Komatsu et al.2010).Our interest in BPG2was sparked because the gene is rapidly repressed upon dark treatment(Kim and von Arnim2006). Notwithstanding that BPG2expression may also be under the control of brassinolide(Komatsu et al.2010),we showed here that BPG2mRNA is regulated by phyto-chrome,since it is inducible by a red light pulse,and this induction is reversed by a far-red light pulse.
We con?rmed the defect in rRNA processing in the bpg2mutant.The rRNA processing is a complex process that is not fully understood(Connolly and Culver2009). Several genes are known to be required for ef?cient rRNA processing in the plastid such as DCL(Bellaoui et al.2003; Bellaoui and Gruissem2004),DAL(Bisanz et al.2003), RNR1(Kishine et al.2004;Bollenbach et al.2005)and PRBP(Park et al.2011).But their molecular mechanisms are elusive.Co-immunoprecipitation of rRNAs with BPG2-YFP(Fig.4b)suggests that BPG2directly contrib-utes to the processing by participating in a ribonucleopro-tein complex that includes the ribosomal RNAs.
The incomplete processing of rRNA is likely to affect the number of effective ribosomes or the ef?ciency of translation in the chloroplast.One of the major proteins that is actively synthesized in the plastid is the D1protein of photosystem II,and the level of D1showed the most striking reduction in the Western blot analysis by Komatsu et al.(2010).Therefore,one can expect signi?cant defects in the functioning of photosystem II in the bpg2mutant. Indeed,several lines of evidence demonstrated conclu-sively that photosystem II activity is compromised at sev-eral places,as indicated by the experimental data presented in our study.In particular,low temperature?uorescence indicated that the energy transfer to the reaction centers of photosystem II is impaired by bpg2mutation.This result, together with reduced accumulation of chlorophylls and b-carotene,may suggest alterations in the PSII antenna system(Ghanotakis et al.1999).Further,the accelerated kinetics of chlorophyll?uorescence after saturating light pulse reduced quantum ef?ciency and energy?ux param-eters,as well as observed de?ciencies in electron transport rate point to defects in the functioning of PSII reaction centers.
Particularly,the ability to perform Q A reduction,as indicated by changes in observed V j and S M values,seems to be affected in bpg2mutant.These results are in agree-ment with the reduced accumulation of D1protein observed previously(Komatsu et al.2010).The presence of an increased pool of defective PSII reaction centers,lack-ing functional D1,may lead to an increased energy dissi-pation in bpg2,similar to damages observed,e.g.,during illumination by excessive light intensities(Pokorska and Romanowska2007).
Considering the dramatic defects in PSII,it is perhaps surprising that two-dimensional BN-PAGE/SDS-PAGE showed only slight changes at the level of Coomassie stained chloroplast proteins(Fig.6b).Some reduction at the level of RbcL and RbcS proteins in the mutant could be found in our result as in Komatsu et al.(2010).But inter-estingly,the ratio between plastid-translated RbcL and cytosolic RbcS within each genotype appears to remain similar.The major difference in higher order complex formation affects the PSI complex,where association between the reaction center,marked by PsaD protein,and its light-harvesting antenna may be altered.A corre-sponding alteration in?uorescence characteristics of PSI manifested itself as a small but signi?cant blue-shift in ?uorescence at77K(Fig.7b).
Light leads to a signi?cant change in the proteome of the etioplast to the proteome of the chloroplast within a short period of time(Kleffmann et al.2007).Although a large
Table1Chlorophyll?uorescence in wild-type(WT)and bpg2mutant plants
F0F M F V/F M V j T1/2(msec)S M
WT46±2.1230±9.98.0±0.020.22a8.0±1.488.5a bpg2168±28450±280.63±0.040.3a0.6±0.269.7a
Average and standard deviation are shown
a Values calculated from averaged?uorescence data
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portion of plastid proteins are synthesized in the cytoplasm, many photosynthetic proteins are encoded by the plastid genome and hence translated by plastid-localized ribo-somes.Therefore,a major change in translational activity is expected in the chloroplast upon illumination.The induction of rRNA production by phytochrome during de-etiolation was described in pea and mustard plastids over40years ago during the dawn of molecular plant physiology(Scott et al.1971;Thien and Schopfer1975). Evidently,the phytochrome-mediated induction of BPG2 expression contributes at least partly to the increased level of correctly processed rRNA to meet the increased demand in ribosomes during the de-etiolation process in chloroplasts.
Acknowledgments Plastid-targeted YFP or CFP fused to the plastid transit peptide of rbcS were kindly provided by Andreas Nebenfu¨hr. We also thank John Dunlap for technical assistance in confocal microscopy and Charles Murphy for help with mass spectrometry. This work was supported by the Division of Chemical Sciences, Geosciences,and Biosciences,Of?ce of Basic Energy Sciences of the U.S.Department of Energy through grant DE-FG02-96ER20223(BK and AGV).PM and AW gratefully acknowledge the support from the Ministry of Science and Higher Education(MNISzW)of the Republic of Poland(grant N303498438)for Figs.5b,7and8. References
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第一节 叶绿素荧光参数及其意义 韩志国，吕中贤（泽泉开放实验室，上海泽泉科技有限公司，上海，200333） 叶绿素荧光技术作为光合作用的经典测量方法，已经成为藻类生理生态研究领域功能最强大、使用最 广泛的技术之一。由于常温常压下叶绿素荧光主要来源于光系统II 的叶绿素a ，而光系统II 处于整个光合 作用过程的最上游，因此包括光反应和暗反应在内的多数光合过程的变化都会反馈给光系统II ，进而引起 叶绿素a 荧光的变化，也就是说几乎所有光合作用过程的变化都可通过叶绿素荧光反映出来。与其它测量 方法相比，叶绿素荧光技术还具有不需破碎细胞、简便、快捷、可靠等特性，因此在国际上得到了广泛的 应用。 1 叶绿素荧光的来源 藻细胞内的叶绿素分子既可以直接捕获光能，也可以间接获取其它捕光色素（如类胡萝卜素）传递来 的能量。叶绿素分子得到能量后，会从基态（低能态）跃迁到激发态（高能态）。根据吸收的能量多少， 叶绿素分子可以跃迁到不同能级的激发态。若叶绿素分子吸收蓝光，则跃迁到较高激发态；若叶绿素分析 吸收红光，则跃迁到最低激发态。处于较高激发态的叶绿素分子很不稳定，会在几百飞秒（fs ，1 fs=10－15 s ）内通过振动弛豫向周围环境辐射热量，回到最低激发态（图1）。而最低激发态的叶绿素分子可以稳定 存在几纳秒（ns ，1 ns=10－9 s ）。 波长吸收荧光红 B 蓝 荧光 热耗散 最低激发态较高激发态基态吸收蓝光吸收红光能量A 图1 叶绿素吸收光能后能级变化（A ）和对应的吸收光谱（B ）（引自韩博平 et al., 2003） 处于最低激发态的叶绿素分子可以通过几种途径（图2）释放能量回到基态(韩博平 et al., 2003; Schreiber, 2004)：1）将能量在一系列叶绿素分子之间传递，最后传递给反应中心叶绿素a ，用于进行光化 学反应；2）以热的形式将能量耗散掉，即非辐射能量耗散（热耗散）；3）放出荧光。这三个途径相互竞 争、此消彼长，往往是具有最大速率的途径处于支配地位。一般而言，叶绿素荧光发生在纳秒级，而光化 学反应发射在皮秒级（ps ，1 ps=10－12 s ），因此在正常生理状态下（室温下），捕光色素吸收的能量主要用 于进行光化学反应，荧光只占约3%～5%(Krause and Weis, 1991; 林世青 et al., 1992)。 在活体细胞内，由于激发能从叶绿素b 到叶绿素a 的传递几乎达到100％的效率，因此基本检测不到 叶绿素b 荧光。在常温常压下，光系统I 的叶绿素a 发出的荧光很弱，基本可以忽略不计，对光系统I 叶 绿素a 荧光的研究要在77 K 的低温下进行。因此，当我们谈到活体叶绿素荧光时，其实指的是来自光系 统II 的叶绿素a 发出的荧光。
1 荧光定义 某些化学物质从外界吸收并储存能量而进入激发态，当其从激发态回到基态时，过剩的能量以电磁辐射的形式放射出去即发光，称之为荧光。可产生荧光的分子或原子在接受能量后引起发光，供能一旦停止，荧光现象随之消失。 2 荧光分类 由化学反应引起的荧光称为化学荧光，由光激发引起的荧光称为光致荧光，课题主要研究光致荧光。按产生荧光的基本微粒不同，荧光可分为原子荧光、X 射线荧光和分子荧光，课题主要研究分子荧光。 3 光致荧光机理 某一波长的光照射在分子上，分子对此光有吸收作用，光能量被分子所吸收，分子具有的能量使分子的能级由最低的基态能级上升至较高的各个激发态的不同振动能级，称为跃迁。分子在各个激发态处于不稳定的状态，并随时在激发态的不同振动能级下降至基态，在下降过程中，分子产生发光现象，此过程为释放能量的过程，即为光致荧光的机理。光致荧光的过程按照时间顺序可分为以下几部分。 分子受激发过程 在波长为10~400nm的紫外区或390~780nm的可见光区，光具有较高的能量，当某一特征波长的光照射分子时，是的分子会吸收此特征波长的光能量，能量由光传递到分子上，此过程为分子受激发过程。分子中的电子会出现跃迁过程，在稳定的基态向不稳定的激发态跃迁。跃迁所需要的能量为跃迁前后两个能级的能量差，即为吸收光的能量。分子跃迁至不稳定的激发态中即为电子激发态分子。 在电子激发态中，存在多重态。多重态表示为2S+1。S为0或1，它表示电子在自转过程中，具有的角动量的代数和。S=0表示所有电子自旋的角动量代数和为0，即所有电子都是自旋配对的，那么2S+1=1，电子所处的激发态为单重态， 用S i 表示，由此可推出，S 即为基态的单重态，S 1 为第一跃迁能级激发态的单重 态，S 2 为第二跃迁能级激发态的单重态。S=1表示电子的自旋方向不能配对，说明电子在跃迁过程中自旋方向有变化，存在不配对的电子为2个，2S+1=3，电子 在激发态中位于第三振动能级，称为三重态，用T i 来表示，T 1 即为第一激发态中 的三重态，T 2 即为第二激发态中的三重态，以此类推。
FluorCam多光谱荧光成像技术（Multi-color FluorCam） 自上世纪90s年代PSI公司首席科学家Nedbal教授与公司总裁Trtilek博士等首次将PAM脉冲调制叶绿素荧光技术与CCD技术结合在一起，成功研制生产FluorCam叶绿素荧光成像系统（Nedbal等，2000）以来，FluorCam叶绿素荧光成像技术得到长足发展和广泛应用，先后有封闭式、开放式（包括标准版和大型版）、便携式叶绿素荧光成像系统，及显微叶绿素荧光成像系统、大型叶绿素荧光成像平台（包括移动式、样带式、XYZ三维扫描式等）等，近些年还进一步发展了PlantScreen植物表型成像分析平台（Phenotyping）（有传送带版、XYZ三维扫描版及野外版等）及多光谱荧光成像技术。 Multi-color FluorCam多光谱荧光成像技术包括多激发光－多光谱荧光成像技术和UV 紫外光激发多光谱荧光成像技术： 1.多激发光－多光谱荧光成像技术：通过光学滤波器技术，仅使特定波长的光（激发光） 到达样品以激发荧光，同时仅使特定波长的激发荧光到达检测器。不同的荧光发色团（如叶绿素或GFP绿色荧光蛋白等）对不同波长的激发光“敏感”并吸收后激发出不同波长的荧光，根据此原理可以选配2个或2个以上的激发光源、绿波轮及相应滤波器，对不同波长荧光（多光谱荧光）进行成像分析。如FluorCam便携式GFP/Chl.荧光成像仪及FluorCam封闭式GFP／Chl.荧光成像系统具备红光和兰光及相应滤波器，可以对GFP和叶绿素荧光成像分析；FluorCam开放式多光谱荧光成像系统可以进一步选配不同颜色的激发光，如除红光、蓝光外，还可选配绿色光源及相应滤波器，以对YFP进行荧光成像分析等； 2.UV紫外光激发多光谱荧光成像技术：长波段UV紫外光（320nm－400nm）对植物叶片 激发，可以产生具有4个特征 性波峰的荧光光谱，4个波峰 的波长为兰光440nm（F440）、 绿光520nm（F520）、红光690nm （F690）和远红外740nm （F740），其中F440和F520 统称为BGF，由表皮及叶肉细 胞壁和叶脉发出，F690和F740 为叶绿素荧光Chl-F。紫外光 激发多光谱荧光（UV-MCF）可 以用来灵敏、特异性地评估植 物生理状态包括受胁迫状态， 包括干旱、病虫害、环境污染、 氮胁迫等 本文就FluorCam多光谱荧光成像技术产品及最新应用案例做一简单介绍，其中FluorCam便携式GFP/Chl荧光成像仪（Handy GFPCam）和FluorCam封闭式GFP/Chl荧光成像系统（Closed GFPCam）已有较为详细的资料介绍，在此不再专门介绍。
叶绿素荧光研究背景知识介绍 前言 近些年来，叶绿素荧光技术已经逐渐成为植物生理生态研究的热门方向。荧光数据是植物光合性能方面的必要研究内容。目前这种趋势由于叶绿素荧光检测仪的改进而得到发展。然而荧光理论和数据解释仍然比较复杂。就我们所了解的情况来看，目前许多研究者对荧光理论不是很清楚，仪器应用仅仅限于简单的数据说明的基础上，本文在此基础上，目的在于简单明晰地介绍相关理论和研究要点，以求简单明确地使用叶绿素荧光检测设备，充分分析实验数据，重点在于植物生理生态学技术的应用和限制。 荧光测量基础 植物叶片所吸收的光的能量有三个走向：光合驱动、热能、叶绿素荧光。三个过程之间存在竞争，其中任何一个效率的增加都将造成另外两个产量的下降。因此，测量叶绿素荧光产量，我们可以获得光化学过程与热耗散的效率的变化信息。尽管叶绿素荧光的总量很小(一般仅占叶片吸收光能总量的1-2%)，测量却非常简单。荧光光谱不同于吸收光谱，其波长更长，因此荧光测量可以通过把叶片经过给定波长的光线的照射，同时测量发射光中波长较长的部分光线的量来实现。有一点需要注意的是，这种测量永远是相对的，因为光线不可避免会有损失。因此，所有分析必须把数据进行标准化处理，包括其进一步计算的许多参数也是如此。 调制荧光仪的出现是荧光研究技术的革命性的创新。在这类仪器中，测量光源是调制(高频率开关)的，其检测器也被调谐来仅仅检测被测量光激发的荧光。因此，相对的荧光产量可以在背景光线(主要是指野外全光照的条件下)存在的条件下进行测量。目前绝大多数的荧光仪采用了调制系统，同时也强烈建议选择调制荧光仪(Kate Maxwell,2000)。 为什么荧光产量会发生改变？Kautsky效应和Beyond 叶绿素荧光产量的变化最早在1960年被Kautsky和其合作者发现。他们发现，当把植物叶片从黑暗中转入光下，荧光产量瞬间上升(大约在1秒左右)这种上升可以解释为光合途径中电子受体的还原(可接受电子的受体的减少)。一旦PSII吸收光能，初级电子受体Q A(质体醌)接受了电子，它将不能再接受电子，直到它把电子传递给下一级电子载体Q B。此期间，反应中心是关闭的，反应中心关闭的比
对于叶绿素荧光全方面的研究 叶绿素荧光现象的发现 将暗适应的绿色植物突然暴露在可见光下后，植物绿色组织发出一种暗红色，强度不断变化的荧光。荧光随时间变化的曲线称为叶绿素荧光诱导动力学曲线。最直观的表现是，叶绿素溶液在透射光下呈绿色，在反射光下呈红色的现象。其本质是，叶绿素吸收光后，激发了捕光色素蛋白复合体，LHC将其能量传递到光系统2或光系统1，期间所吸收的光能有所损失，大约3%-9%的所吸收的光能被重新发射出来，其波长较长，即叶绿素荧光。 叶绿素荧光动力学研究的特点 1、叶绿素荧光动力学特性包含着光合作用过程的丰富信息 光能的吸收和转换 能量的传递与分配 反应中心的状态 过剩光能及其耗散 光合作用光抑制与光破坏 2、可以对光合器官进行“无损伤探查” 3、操作步骤简单快捷 光合作用的光抑制 光抑制是过剩光能造成光合功能下降的过程。过剩光能指植物所吸收的光能超出光化学反应所能利用的部分。过去人们把光抑制与光破坏等同起来，认为发生了光抑制就意味着光和机构遭到破坏。甚至把光抑制、光破坏、光氧化等，沦为一体。 光抑制的基本特征表现为： 光合效率下降说明叶片吸收的光能不能有效地转化为化学能。光破坏：PSII 是光破坏的主要场所，破坏也可能发生在反应中心也可能发生在与次级电子受体结合的蛋白上。发生光破坏后的结果：电子传递受阻、光合效率下降。当过剩的光能，不能及时有效地排散时，会对光合机构造成不可逆的伤害，如光氧化、光漂白等等。一切影响二氧化碳同化的外界因素，如低温、高温、水分亏缺、矿质元素亏缺等都会减少对光能的利用，导致过剩光能增加，进而加重光破坏。 植物防御破坏的措施 1、减少对光能的吸收 增加叶片的绒毛、蜡质 减少叶片与主茎夹角 2、增强代谢能力 碳同化 光呼吸 氮代谢 3、增加热耗散 依赖叶黄素循环的热耗散 状态转换 作用中心可逆失活 光合作用
植物表型组学研究技术（一） ——FluorCam叶绿素荧光成像技术
FluorCam叶绿素荧光成像技术 Rousseau等（High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis.Plant Methods, 2013, 9:17），利用FluorCam开放式叶绿素荧光成像系统作为高通量表型分析平台，采用图像阈值分割等分析方法，对植物病原体感染进行了定量分析检测，根据Fv/Fm将感染分为不同阶段／等级，特别是可以将用其它方法难以分辨出来的感染前期加以分辨，并对5个品种的菜豆对普通细菌性疫病的抗性进行了定量分析评价。 PSI公司首席科学家Nedbal教授与公司总裁Trtilek博士等首次将PAM叶绿素荧光技术（Pulse Amplitude Modulated technique—— 脉冲调制技术）与CCD技术结合在一起，于1996 年在世界上成功研制生产出FluorCam叶绿素荧 光成像系统（Heck等，1999；Nedbal等，2000； Govindjee and Nedbal, 2000）。FluorCam叶 绿素荧光成像技术成为上世纪90年代叶绿素荧 光技术的重要突破，使科学家对光合作用与叶 绿素荧光的研究一下子进入二维世界和显微世 界，广泛应用于植物生理生态、植物胁迫与抗 性监测、作物育种、植物表型分析等。不同于 其它成像分析技术，FluorCam叶绿素荧光成像 只对叶绿素荧光波段敏感，可以有效避免环境 光的干扰，特异性、高灵敏度反映植物生理生 态状况。 主要功能特点如下： 1)高灵敏度CCD，时间分辨率可达50帧／秒，有效抓取叶绿素荧光瞬变；可选配高分 辨率CCD，分辨率1392x1040像素，用于气孔功能成像分析、稳态荧光如GFP荧光测量等
藻类研究监测快讯 藻类是水体生态系统中的生产者，在生态系统中起着不可或缺的作用。随着能源与环境方面研究的深入，藻类已经越来越多的被利用到实验当中。叶绿素荧光是藻细胞中的叶绿素吸收光能后受激发而释放出的能量，通过检测荧光的强弱， 可初步判断藻类的光合作用强度及生理状况。该项技术使藻 类的生理生化研究变得更加简单、方便、精确。 重要参数如下： Ft瞬时荧光，与藻细胞浓度、叶绿素浓度有 关。在暗适应状态下测得的Ft值即为Fo最小荧 光值，在给予饱和光照时，即为Fm最大荧光值； QY反映藻类的光合效率，对胁迫非常敏感；暗适应条件下测得的QY值为最大光合效率值即(Fm-Fo)/Fm，反映藻类的潜在光合效率，光照下测得的QY值为有效光量子产量即(Fm’-Ft)/Fm’，反映藻类的实际光合效率。 OJIP曲线快速荧光诱导曲线，可测定藻类在由暗适应转到光照下的瞬间荧光变化，其中 FixArea与藻类叶绿素浓度 呈正相关，可作为藻类浓度 指标；PI为功能指数，对 胁迫非常敏感。有些胁迫不 会影响PSⅡ，也不会导致 QY降低，但可通过PI体 现出来，PI可以反映三个方面：反应中心密度、用于电荷分离过程的光子吸收率、电子传递效率。 NPQ 非光化学荧光淬灭，多余辐射能的散失，反映的藻类的光保护能力。 1、AquaPen探头式藻类荧光仪 AquaPen探头式藻类荧光仪用于水体微藻类的荧光测量，其高灵敏度和便携性可以对水 体较低浓度的浮游植物进行快速测量。检测极限可达0.5 μg Chl/L,测量计算参数：Fo, Ft, Fm, Fm‘, QY, OJIP, NPQ等。 光化学光和饱和光的强度在0 - 3,000 μmol·m-2·s-1可调，光 化光的持续时间可调，界面简单，易于操作，内存可达4Mb， 4节AAA电池供电，数据可通过USB数据线传至计算机或 掌上电脑。检测器前带有暗适应罩子，适合野外测量。
Fluorcam荧光成像技术及其在光合作用研究 中的应用 Eco‐lab生态实验室 北京易科泰生态技术有限公司 info@eco‐https://www.doczj.com/doc/1f15966837.html,
目录 1、叶绿素荧光成像技术发展过程 2、荧光参数及其生理意义 3、PSI介绍（荧光成像的发明者） 4、PSI产品介绍 5、应用案例
叶绿素荧光技术发展历程 ?Kautsky effect: Kautsky and Hirsch(1931)首次用肉眼发现叶绿素荧光现象并发表论文“CO2同化新实验”，后被称作“Kautsky effect” ?PAM(Pulse Amplitude Modulated Fluorometer): Schreiber（1986）等发明了PAM脉冲调制技术测量叶绿素荧光。?FluorCam：KineKc imaging of chlorophyll fluorescence: Ladislav Nedbal（2000）等于上世纪90年代末期发明了与 PAM技术相结合的叶绿素荧光成像技术
成像测量局部放大
荧光参数及其意义 ?Fo、Fm与QY，此外还有PAR_Abs及ETR ?Kautsky诱导效应：Fo，Fp，Fv，Ft_Lss，QY，Rfd ?荧光淬灭分析：Fo，Fm，Fp，Fs，Fv，QY，NPQ，Qp，Rfd 等50多个参数 ?OJIP曲线：快速荧光诱导曲线。Fo，Fj，Fi，P或Fm，Mo（OJIP曲线初始斜率）、FixArea固定面积、Sm（对关闭所有光反应中心所需能量的量度）、QY、PI等 ?LC光响应曲线：Fo，Fm，QY，QY_Ln
叶绿素荧光仪著名厂商 ?PSI：捷克布尔诺Brno（孟德尔在此发现著名的孟德尔遗传定律），Ladislav Nedbal为首席科学家和主要股东（另一股东为David Kramer，美国密执根州立大学教授），1997年为美国华盛顿大学H.Pakrasi教授研制成了第一台FluorCam荧光成像系统。主要产品有： –FluorCam叶绿素荧光成像系列产品 –FL3500/FL5000双调制荧光仪系列产品 –FluorPen及AquaPen等手持式荧光仪产品 –光养生物反应器等藻类培养与在线监测产品 –光源与植物培养室 ?Optics：美国，主要产品为OS5p‐PAM叶绿素荧光仪等?Walz：德国，主要产品为PAM2500叶绿素荧光仪等
荧光分析法的特点及在环境分析中的应用 摘要：论文综述了荧光分析法的特点及在环境分析中的应用。重点分析了荧光分析法的原理、特点，以及常用的荧光分析法的讨论。分析了荧光分析法在环境监测中的应用，测定范围和发展情况。 关键词：荧光分析；环境分析；应用 1.引言 环境中分析、监测的对象往往是微量、超微量的物质，有很多还具有时间性和空间性，因此对分析技术要求越来越高。荧光分析法和分光光度法以其灵敏度高、检测限低、准确性好等优点在近年来得到了迅速发展。荧光分子探针的设计合成以及荧光分析法在环境分析化学中的应用是方兴未艾的研究方向[1]。 分子荧光分析具有检测限低，灵敏度高，选择性好，取样量少，方法简捷快速等特点，是一种重要的光谱化学分析手段，其中荧光分子探针检测技术在环境分析化学中占有重要的地位[2]。因此，在对环境的分析中，荧光分析法应用非常广泛，从天然水、饮用水到废水、污水；从土壤、大气到动植物；从人的头发、骨骼、血液到内脏等各个器官，涉及到的样品和应用范围几乎无所不有[3]。 2.荧光分析法的原理和特点 2.1.荧光分析法 2.1.1荧光及荧光分析 荧光是荧光化合物在受到紫外光、电和化学等能量激发后，电子从基态跃迁到激发态，然后通过辐射衰变释放出光子而回复到基态，即产生荧光。这些物质会在极短的时间内(8-10秒)发射出各种颜色和不同强度的可见光，而当紫外光停止照射时，所发射的光线也随之很快地消失。 荧光分析是指利用某些物质在紫外光照射下产生荧光的特性及其强度进行物质的定性和定量的分析的方法。1852年G.G.斯托克斯(G.G.Strokes)发现荧光，真正的荧光光谱测量则始于本世纪60年代。 2.1.2荧光激发光谱和发射光谱 荧光是一种光致发光现象，由于分子对光的选择性吸收，不同波长的入射光便具有不同的激发效率。如果固定荧光的发射波长不断改变激发光的波长，并记
1荧光定义 某些化学物质从外界吸收并储存能量而进入激发态，当其从激发态回到基态时，过剩的能量以电磁辐射的形式放射出去即发光，称之为荧光。可产生荧光的分子或原子在接受能量后引起发光，供能一旦停止，荧光现象随之消失。 2荧光分类 由化学反应引起的荧光称为化学荧光，由光激发引起的荧光称为光致荧光，课题主要研究光致荧光。按产生荧光的基本微粒不同，荧光可分为原子荧光、X 射线荧光和分子荧光，课题主要研究分子荧光。 3光致荧光机理 某一波长的光照射在分子上，分子对此光有吸收作用，光能量被分子所吸收，分子具有的能量使分子的能级由最低的基态能级上升至较高的各个激发态的不同振动能级，称为跃迁。分子在各个激发态处于不稳定的状态，并随时在激发态的不同振动能级下降至基态，在下降过程中，分子产生发光现象，此过程为释放能量的过程，即为光致荧光的机理。光致荧光的过程按照时间顺序可分为以下几部分。 3.1 分子受激发过程 在波长为10~400nm的紫外区或390~780nm的可见光区，光具有较高的能量，当某一特征波长的光照射分子时，是的分子会吸收此特征波长的光能量，能量由光传递到分子上，此过程为分子受激发过程。分子中的电子会出现跃迁过程，在稳定的基态向不稳定的激发态跃迁。跃迁所需要的能量为跃迁前后两个能级的能量差，即为吸收光的能量。分子跃迁至不稳定的激发态中即为电子激发态分子。 在电子激发态中，存在多重态。多重态表示为2S+1 o S为0或1,它表示电子在自转过程中，具有的角动量的代数和。S=0 表示所有电子自旋的角动量代数和为0,即所有电子都是自旋配对的，那么2S+仁1,电子所处的激发态为单重态，用S i 表示，由此可推出，S0 即为基态的单重态，S1 为第一跃迁能级激发态的单重态，S2为第二跃迁能级激发态的单重态。S=1表示电子的自旋方向不能配对，说明电子在跃迁过程中自旋方向有变化，存在不配对的电子为2个，2S+仁3,电子在激发态中位于第三振动能级，称为三重态，用T i 来表示，T1 即为第一激发 态中的三重态，T2即为第二激发态中的三重态，以此类推。 分子跃迁至各个激发态中，状态不稳定，随时会释放出能量，释放能量的类型有两种：一种是辐射跃迁，另一种是非辐射跃迁，释放能量会回到稳定的基态。
园艺学报，（）：– 2014411122152224 http: // www. ahs. ac. cn Acta Horticulturae Sinica E-mail: yuanyixuebao@https://www.doczj.com/doc/1f15966837.html, 收稿日期：2014–08–22；修回日期：2014–10–24 基金项目：河北省海外高层次人才百人计划项目（E2013100011）；河北省杰出青年科学基金项目（C2013204118）；‘十二五’农村领域国家科技计划课题（2012AA100202-5）；农业部农业科研杰出人才培养计划项目（2130106）；高等学校博士学科点专项基金项目（20121302110006） 大白菜叶色突变体的HRM 鉴定及其叶绿素荧光参数分析 刘梦洋，卢 银，赵建军，王彦华，申书兴* （河北农业大学园艺学院，河北省蔬菜种质创新与利用重点实验室，河北保定 071000） 摘 要：将大白菜经甲基磺酸乙酯（EMS ）诱变种子获得的42株叶色突变体按照生殖时期叶片颜色和叶绿素含量分为9种类型：深绿色、灰绿色、绿色、浅绿色、白绿色、白浅绿色、黄绿色、黄浅绿色、黄色；利用高分辨率熔解曲线（high resolution melting ，HRM ）技术对叶绿素荧光基因HCF164突变进行了筛选并结合叶绿素荧光参数测定，获得了1株黄绿色高光合效率突变体A29，1株黄绿色光合结构损伤突变体A35和1株浅绿色光合电子传递受阻突变体A21；对另外7个叶色相关基因的突变进行了HRM 鉴定，表明叶绿素相关基因ATRCCR 、CLH2、PORA 突变可能是造成18个突变体叶色变化的主要原因，黄叶特异基因家族YLS 突变与叶色变化也有关系。 关键词：大白菜；诱变；突变体叶色；HRM ；叶绿素荧光 中图分类号：S 634.1 文献标志码：A 文章编号：0513-353X （2014）11-2215-10 HRM Identification and Chlorophyll Fluorescence Characteristics on Leaf Color Mutants in Chinese Cabbage LIU Meng-yang ，LU Yin ，ZHAO Jian-jun ，WANG Yan-hua ，and SHEN Shu-xing * （College of Horticulture ，Agricultural University of Hebei ，Key Laboratory for Vegetable Germplasm Enhancement and Utilization of Hebei ，Baoding ，Heibei 071001，China ） Abstract ：Forty-two leaf color mutants of Chinese cabbage obtained through EMS seeds mutagenesis were used as materials in this study. According to leaf color and leaf chlorophyll content at generative growth mutations were suggested to be divided into 9 types ：Dark green ，gray-green ，green ，light green ，white-green ，light white-green ，yellow-green ，light yellow-green and yellow. By detecting the nucleotide variation of the gene HCF164 related to chlorophyll fluorescence using HRM technology and by measuring chlorophyll fluorescence characteristics ，we identified one yellow-green leaf color mutant A29 with high photosynthesis efficiency ，one yellow-green leaf color mutant A35 with photosynthetic structure damages ，one light green mutant A21 with photosynthetic electron transport obstruction. Through identifying other 7 leaf-color-related genes by HRM ，mutation of chlorophyll-related genes ATRCCR ，CLH2 and PORA could be the main reason resulted in 18 leaf color mutants ，mutation of yellow-leaf- specific genes was also affected the variation of leaf color. * 通信作者 Author for correspondence （E-mail ：shensx@https://www.doczj.com/doc/1f15966837.html, ）
1.1 时间分辨荧光分析技术 时间分辨荧光生化分析技术是基于稀土荧光配合物特殊的荧光性质而建立起来的，自1978年提出以来[1]，已广泛的应用于免疫分析、核酸测定、荧光显微镜成像、细胞识别、单细胞原位测定、生物芯片等生化领域，并发展出了相应的时间分辨荧光免疫测定法、时间分辨荧光DNA 杂交测定法、时间分辨荧光显微镜成像测定法、时间分辨荧光细胞活性测定法及时间分辨荧光生物芯片测定法等分支。 本节主要对稀土荧光配合物的发光机理、荧光性质，时间分辨荧光测定的原理，时间分辨荧光免疫分析技术，时间分辨荧光显微镜成像技术的研究进展等加以介绍。 1.1.1 稀土荧光配合物的发光机理及荧光性质 稀土元素指的是元素周期表中IIIB 族的镧系元素以及钪和钇，共17种元素。其中镧系元素的外层电子结构为4f 0-145d 0-106s 1-2，由于5s 和5p 电子对4f 电子的屏蔽作用，导致这些金属及其离子的性质十分相似。图1.1给出了四种三价稀土离子的基态及激发态电子能级图[2]。 1020 152530355 E N E R G Y ,103c m -1 6 H 5/2 G 5/2 6 H 15/2 7 F 0 F 2D 0 5D 1 7F 6 F 5 4 5D 3 13/2 4 9/2 Sm 3+ Eu 3+ Tb 3+ Dy 3+ H 9/2 图1.1 部分三价稀土离子的电子能级图 Fig. 1.1 Electronic energy levels of certain lanthanide(III) ions 大部分稀土离子本身是不具有荧光性质的，只有Sm 3+、Eu 3+、Tb 3+和Dy 3+的水溶液在紫外光或可见光的激发下能够发出微弱的荧光。当Sm 3+、Eu 3+、Tb 3+和Dy 3+与某些有机配位体形成配合物时其荧光强度会显著增强，这种发光是基于配合物由配位体到中心稀土离子的能量转移所产生的[3-8]。以铕(III)配合物为例，其荧
Sensors2011, 11, 3765-3779; doi:10.3390/s110403765 OPEN ACCESS sensors ISSN 1424-8220 https://www.doczj.com/doc/1f15966837.html,/journal/sensors Article Hyperspectral and Chlorophyll Fluorescence Imaging to Analyse the Impact of Fusarium culmorum on the Photosynthetic Integrity of Infected Wheat Ears Elke Bauriegel 1,*, Antje Giebel 1 and Werner B. Herppich 2 1Department of Engineering for Crop Production, Leibniz-Institute for Agricultural Engineering Potsdam-Bornim, D-14469 Potsdam, Germany; E-Mail: agiebel@atb-potsdam.de 2Department of Horticultural Engineering, Leibniz-Institute for Agricultural Engineering Potsdam-Bornim, D-14469 Potsdam, Germany; E-Mail: wherppich@atb-potsdam.de * Author to whom correspondence should be addressed; E-Mail: ebauriegel@atb-potsdam.de; Tel.: +49-331-5699-414; Fax: +49-331-5699-849. Received: 24 January 2011; in revised form: 23 March 2011 / Accepted: 25 March 2011 / Published: 28 March 2011 Abstract: Head blight on wheat, caused by Fusarium spp., is a serious problem for both farmers and food production due to the concomitant production of highly toxic mycotoxins in infected cereals. For selective mycotoxin analyses, information about the on-field status of infestation would be helpful. Early symptom detection directly on ears, together with the corresponding geographic position, would be important for selective harvesting. Hence, the capabilities of various digital imaging methods to detect head blight disease on winter wheat were tested. Time series of images of healthy and artificially Fusarium-infected ears were recorded with a laboratory hyperspectral imaging system (wavelength range: 400 nm to 1,000 nm). Disease-specific spectral signatures were evaluated with an imaging software. Applying the ?Spectral Angle Mapper‘ me thod, healthy and infected ear tissue could be clearly classified. Simultaneously, chlorophyll fluorescence imaging of healthy and infected ears, and visual rating of the severity of disease was performed. Between six and eleven days after artificial inoculation, photosynthetic efficiency of infected compared to healthy ears decreased. The severity of disease highly correlated with photosynthetic efficiency. Above an infection limit of 5% severity of disease, chlorophyll fluorescence imaging reliably recognised infected ears. With this technique, differentiation of the severity of disease was successful in steps of 10%. Depending on the quality of chosen regions of interests, hyperspectral imaging readily detects head blight 7 d after inoculation
叶片荧光测量实验报告 1.实验目的 2.实验方法 利用PAM100,荧光成像系统测量叶绿素荧光 3.实验原理及一些参数的意义 荧光的变化反映光合与热耗散的变化。 光化学淬灭（Photochemical Quenching）:由于光合作用引起的荧光下降，反映了光合活性的高低。 qP=(Fm’-Fs)/Fv’=1-(Fs-Fo’)/(Fm’-Fo’) （基于“沼泽模型”） qL=(Fm’-F)/(Fm’-Fo’)·Fo’/F=qP·Fo’/F （基于“湖泊模型”） 非光化学淬灭（Non-Photochemical Quenching）:由于热耗散引起的荧光下降。 qN=(Fv-Fv’)/Fv=1-(Fm’-Fo’)/(Fm-Fo) NPQ=(Fm-Fm’)/Fm’=Fm/Fm’-1 ，不需测定Fo’，适合野外调查qN或NPQ反映了植物耗散过剩光能转化为热的能力，反映了植物的光保护能力。 Fv/Fm =(Fm-Fo)/Fm : PS II的最大量子效率，反映植物潜在最大光合能力，高等植物一般在0.8－0.84之间，当植物受到胁迫（Stress）时，Fv/Fm显著下降。 ΦPS II = Yield = (Fm’-Fs)/Fm’ = ΔF/Fm’= qP·Fv’/Fm’: 任一光照状态下PS II的实际量子产量（实际光合能力、实际光合效率）
不需暗适应，不需测定Fo’，适合野外调查。 Y(NPQ)=1-Y(II)-1/(NPQ+1+qL(Fm/Fo-1))：调节性能量耗散，PS II 处调节性能量耗散的量子产量。若Y(NPQ)较高，一方面表明植物接受的光强过剩，另一方面则说明植物仍可以通过调节（如将过剩光能耗散为热）来保护自身。Y（NPQ）是光保护的重要指标。 Y(NO)=1/(NPQ+1+qL(Fm/Fo-1))：非调节性能量耗散 PS II处非调节性能量耗散的量子产量。若Y(NO)较高，则表明光化学能量转换和保护性的调节机制（如热耗散）不足以将植物吸收的光能完全消耗掉。也就是说，入射光强超过了植物能接受的程度。这时，植物可能已经受到损伤，或者（尽管还未受到损伤）继续照光的话植物将要受到损伤。Y（NO）是光损伤的重要指标。 P：光合速率，即相对电子传递速率rETR Pm: 最大光合速率，即最大相对电子传递速率rETRmax α：初始斜率，反映了光能的利用效率 β：光抑制参数 Ik=Pm/α：半饱和光强，反映了样品对强光的耐受能力。
第四章 叶绿素荧光技术应用 第一节 叶绿素荧光参数及其意义 韩志国，吕中贤（泽泉开放实验室，上海泽泉科技有限公司，上海，200333） 叶绿素荧光技术作为光合作用的经典测量方法，已经成为藻类生理生态研究领域功能最强大、使用最广泛的技术之一。由于常温常压下叶绿素荧光主要来源于光系统 II 的叶绿素 a ，而光系统 II 处于整个光合作用过程的最上游，因此包括光反应和暗反应在内的多数光合过程的变化都会反馈给光系统 II ，进而引起叶绿素 a 荧光的变化，也就是说几乎所有光合作用过程的变化都可通过叶绿素荧光反映出来。与其它测量方法相比，叶绿素荧光技术还具有不需破碎细胞、简便、快捷、可靠等特性，因此在国际上得到了广泛的应用。 1 叶绿素荧光的来源 藻细胞内的叶绿素分子既可以直接捕获光能，也可以间接获取其它捕光色素（如类胡萝卜素）传递来的能量。叶绿素分子得到能量后，会从基态（低能态）跃迁到激发态（高能态）。根据吸收的能量多少，叶绿素分子可以跃迁到不同能级的激发态。若叶绿素分子吸收蓝光，则跃迁到较高激发态；若叶绿素分析吸收红光，则跃迁到最低激发态。处于较高激发态的叶绿素分子很不稳定，会在几百飞秒（fs ，1 fs=10－15 s ）内通过振动弛豫向周围环境辐射热量，回到最低激发态（图 1）。而最低激发态的叶绿素分 子可以稳定存在几纳秒（ns ，1 ns=10－9 s ）。 A 较高激发态 B 热耗散 吸收蓝光 吸收红光 最低激发态 能量 荧光 基态 蓝 波长 红 荧光 图 1 叶绿素吸收光能后能级变化（A ）和对应的吸收光谱（B ）（引自韩博平 et al., 2003） 处于最低激发态的叶绿素分子可以通过几种途径（图 2）释放能量回到基态(韩博平 et al., 2003; Schreiber, 2004)：1）将能量在一系列叶绿素分子之间传递，最后传递给反应中心叶绿素 a ，用于进行光化学反应；2）以热的形式将能量耗散掉，即非辐射能量耗散（热耗散）；3）放出荧光。这三个途径相互竞争、此消彼长，往往是具有最大速率的途径处于支配地位。一般而言，叶绿素荧光发生在纳秒级，而 光化学反应发射在皮秒级（ps ，1 ps=10－12 s ），因此在正常生理状态下（室温下），捕光色素吸收的能 量主要用于进行光化学反应，荧光只占约 3%～5%(Krause and Weis, 1991; 林世青 et al., 1992)。 在活体细胞内，由于激发能从叶绿素 b 到叶绿素 a 的传递几乎达到 100％的效率，因此基本检测不到叶绿素 b 荧光。在常温常压下，光系统 I 的叶绿素 a 发出的荧光很弱，基本可以忽略不计，对光系统 I 叶绿素 a 荧光的研究要在 77 K 的低温下进行。因此，当我们谈到活体叶绿素荧光时，其实指的是来自光系统 II 的叶绿素 a 发出的荧光。
PlantScreen 一、植物表型组学与PlantScreen 植物表型成像分析技术 自 20 世纪 90 年代初以来，生命科学领域出现了最为引人注目的“组学”新概念和新学科，如基因组学（genomics ）、转录组学（transcriptomics ）、蛋白质组学（proteomics ）和代谢组学（metabolomics ）等。伴随各种组学的不断兴起和发展，90年代末，人们提出了表型组（phenome ）和表型组学（phenomics ）的概念。2013年Monya Baker 在《Nature 》发表文章“THE ‘OMES PUZZLE ”将表型组学称为“前景光明（Aspiring ）”的组学研究项目[1]。 表型组定义为：在细胞、组织、器官、生物体或 种属水平上表现出的所有表型的组合。表型组学可定 义为一门在基因组水平上系统研究某一生物或细胞 在各种不同环境条件下所有表型的学科[2] DNA 芯片技术的进一步完善，为植物功能基因 组学研究提供契机[3]。而之前植物表型组学一直缺乏 合适的研究仪器，研究者不得不使用传统方法来获取 表型组学的海量数据。随着近几年FluorCam 叶绿素 荧光成像技术、RGB 彩色成像分析技术乃至集合了 多种最先进表型成像分析技术和植物自动培养技术 的PlantScreen 植物表型成像分析技术逐渐成熟，直 接促进了植物表型组学的发展，同时为基因组、蛋白 组、代谢组及表型组数据进一步整合起来研究提供了 新的挑战和可行性。关于FluorCam 叶绿素荧光成像 技术与RGB 彩色成像分析技术在表型组学中的应用， 请见： 植物表型组学研究技术（一）——FluorCam 叶 绿素荧光成像技术 植物表型组学研究技术（二）——叶绿素荧光成 像与 RGB 彩色成像分析系统 PSI 公司在功能强大的FluorCam 叶绿素荧光成像技术基础上，结合LED 植物智能培养、自动化控制系统、植物热成像分析、植物近红外成像分析、植物高光谱分析、自动条码识别管理、RGB 真彩 3D 成像、自动称重与浇灌系统等多项先进植物表型技术，开发出了PlantScreen 植物表型成像分析系统。这一大型系统切合国际最新的植物表型组学研究，以最优化的方式实现了拟南芥、小麦、水稻、玉米乃至 目前各种热门与不热门的组学项目，表型组(phenome)被认为是“前景极为光明的”[1]