The 520-nm absorption change in barley and a chlorophyll b-deficient mutant

The

520-nm

Absorption

Change b-Deficient

WILLIA;\I

Ieeceivcd

JIIIE

in Barley

and

a Chlorophyll

Mutant

W. HILDlLI~;‘I‘H’

-L, lM9;

nccept~ctl

April

6, 1070

Normnl I)arlry leaf :ttld :t chloroph,vll h-dcfic*icanl I~II~:~III shl)w similar flash-illtlllced c~h:tngcs ai 520 11m, most prohahly dr1c1 to c:trotc?llc)ids r:ltht:r 1ha11 c~hlorophyll h. The foll~~wi~~g :I L-swit.chcd laser flash, arc rapid :urtl illtermediatr rcccjvery ph:tsps, similar ill the lrormal :tnd 111111ani, I)arley Icaf. The fill:d rr’covcry phase is 50’ ; slower in the rnrltant. barley. The(:ytochromef oxidation rate is two timesfastcr in the mutant than in the xlormal barlcy leaf. The sensitivity of t)hc 5%11m recsovery phases to ternperat ,1rc decrewsc indic:tt,cs a rclatiotlship of the 52%ILIIL absorption change with t,he state of the clhoroplast mcml)r,ztlc. Further tlnt:t :tro prrscntc~tl OII extinction POelliCklts itlltl clllant,lim !?rltl.

The absorptiotk incrtt:~w occurring at X0 11111in green plants and algnr: (1) has bccw attributed in part to chlorophyll h (Z-4). This identification wxs b:wod on absorption ch:mges near 648 nm which were prwumabl~ correl:Lted xvith the total absorption change at 520 nm. Altornativel~-, indirect evidcncc that th(l X0-nm spectral shift, is due to carotcnoids NW reported by Chance and co-workers (,T, 6). Their proposal IV:LSbased on studiw of a 8-c:Lrotr~rlc-deficient mutant or Chlnlrrllt/fJlt,orras reinharr/ii. This pnl~~green mutant, I\-it11 a chl a to chl 11ratio of 3 (7), she\\-s no .X0-Iun absorption change, char:rct,wistit of the wiltltypc~ Chlaw?/flO?,lOJ~GS. Addition:d evidence in favor of the latter hypothwis c:~m~ from the study of a chlorophyll b-d&ciclnt’ mutant of barIcy. This mutant,, with :I narly normal content of &carotene, sho\vs a .X0-nm absorption iItcrease urlder continuous illumination (S). The prcwnt work tl(~monstr:ttrs :L flashintluwd absorption change at 520 nm, with thrw dist,inct recowry phasw, in both the normal and mutant barley. A\lso, tht> tempcr:lturc~ d(~pr~ndencr: of the X0-nm recovcq 1 Stlpportrtl Copyright

1)~ lJ.S.P.1I.S. @ 1970 1)~. Academic

(:rallt

5TlG51277.O(j. Press,

ln~.

phuses is describr,d, as well as the cyt,ochrome j oxid:ltiorl-reduction kinetics. A prelimimlry rqwrt’ has been given elsewhere (9). MATI<:l: ri\l,s ANI) MIGTIIOl)S The tlorm:tl :ultl matalit strains of b:trlt:y2 (flordeunz v~~l~qurc) were groan ill a greenhouse, harvcst~d, and rlsetl immediately for experiments. Only intact, IWVCS w(trc II& itI thcsc experimellts, due to the small sr~ppl,v of mntnnt barley. The pigment, contrlrt of 1hc lrormal and mrrt ant barley W:LS determilled by T. IIiy:tma (7), and is shown in Table I. The appxr:a,tus for measllring rapid flash-intlr~ced ahsorpt ion ch:t~ges in photosynthetic s:tml)les WARS developed t>y I )CVitlIlt :irld Chance (11, 12). Briefly, this tjcchniquc rmploys n Q-switched laser pldae (pulse length -30 nsec) reflected on to a 1.5 X 1.5-cm2 leaf sample. Three lengths of barley leaf nrc placed side-hy-side with overlap, a11t1 pressed hrtaecn two lrlcitr: plates. The laser WRV(‘letlgth is fi9-l IIn), wiih nlr itlcident intel,sity of IO”10’ W/m12 (mcnsllred with R TR(; holomctrr), varied hy mritlx3 r,f :I rlcgativc lcks and nellt ml detisity filters. The fiash-indllceti ahsorption changes arc ~RHStired by means of :I single-beam spectrophotomctrr (spectral width = 3-4 nm). The monochro-_____ 2 Swtis kilrdly sllpplied hy Prof. H. lt. Highkin (10).

TABLF:

Pigment

Normal”

I

~lutant

-

Chlorophyll Chlorophyll &Carotene I,utein Neoxanthin

a 6

5900 1800 180 110 3i

II For method of determilling see Ref. 7. h Units of *g/g dry weight,

4Goo 0 110 12 5 pigmerIt,

Ratio Kormal/ mutant

1.3 1”1G 9.2 7.4 coIltent,,

mator lamp can operate at, either llorrnxl voltage, or pulsed overvoltage, to give measllring beam itltensities of -0.03 mW/cm2 or -0.1 mW/cm2, respectively, at 520 nm. The pIllsed Ittrnp voltage gives a constant intensity for -30 msec duritlg which time the rapid killet.ics arc recorded (cf. Fig. JA, B, I), Ii>). The photolnult.iplier (15MI 9524B) is guarded from t,he laser flash by a Corning 4-9G blue-green filter. The outpklt, signal is amplified, balanced by a d-c offset voltage, and displayed on an oscilloscope. For low-temperature studies, an alrmlitlrun fill projects below the sample holder illto an icr-water or liquid nitrogen bath within a11 mrsilvercd Dcwar flask. The temperature is recorded on a Keithley microvoltmeter with a Ctl-rolrstantell thermocouple in contact with the tIpper edge of the leaf sample. A suitable range of low tempera1 llres call be obtained by varying the dept,h of immersion of t,he aluminum fin in t,he liquid nitrogen bath. The dat,a shown in Table II were measured directly from Xerox copies of the photographed oscilloscope traces, after hand-smoot,hirrg of the traces. Each room temperature halftime is an average of 20-30 traces, each 100°K halftime is an average of 10-20 traces, and the tabulated unrertainties are standard deviations. The chlorophyll content of the barley a11d ILIIItant leaves was determilked by crltting a given sample into small pieces, and irrscrtillg in 80y0 test tube, for acetone at Q”, in a glass-stoppered 24 hr. The sample was then macerated wit,h a glass rod and centrifuged. From the optical drnsit.ies at 045 and GG3 nm, the chlorophyll content was ca-

crllated (13). 11EHULTS

Laser-iducetl kinetics. The flash-induced absorption change at 520 nm recovws in three distinct phases, for both the normal

(Fig. lA, B, and C) xrd mutant barley (Fig. lD, E, and F). The rise in halftime for the 5Wnm absorption changes is less than 1 ~scc, measured from recordings five times faster than E’ig. 1A and D. The rapid (R) recovery phase is similar for both the normal (P’ig. 1A) and mutant barley (1;ig. 11~). (R - tli? = (i-7 ~scc.) The intermediate (I) phase is similar in both types of leaves, and occurs as an additional absorption increase (I’ig. 1B and E, I - fl:% = 4 mscc). By comparison, the I-phase occurs as XI absorption increase in Chlorella, as a plateau in spinach leaf, and as a partial recovery in spinach chloroplasts (14, 15). The final (S) recovery phase is slower in the mutant barley (Fig. IF, S - tljz = 1.50 mscc) than in the normal leaf (Fig. lC, ~5’- tl/z = 100 msrc). This difference may bc statistically significant, as shown by the uncertainties tabulated in Table II. The S phuw recovers with t,he tabulated half-times only aftw four or more laser flashes. After the first laser flash or1 a giwn sample, the X phase halftime is 50 msecfor the normal leaf, and 120 msec for the mutant leaf. This slowing of the fi1~1 recovery rate may be due to rxhaust’ion of endogenous substrate after flash activation. Exposure of the leaf to dim white light and CO2 restores the original X20-rim S-phase recovery rate (set also Ref. 15). Roolrz-tel,zpe,^atur,e spectra. Spectra of the M phase for the normal and mutant leaf are shown in Pig. 2. The R-phase spectra are measured from the maximum AOD after the laser flash 011 the time scale of Fig. 1A and 11. The negative peak is shifted from 475 to 4% nm for the mutant leaf, a statistically significant effect. Ko cytochrome f alpha band is observed on this rapid time scale. Spectra of the I phase are shown in E’ig. 3 for the normal and mutant leaf. The Iphase spectra are measured from the steadystate AOD on the time scale of Fig. 1A and D (or the initial AOD on Fig. 1B and E). A shoulder appears near 535 nm for both spectra. The negative peak is again shifted from 475 to 486 nm for the mutant leaf (cf. Ref. S). On this time scale, cytochrome f is fully oxidized, with a spectral peak at 5.54 nm. The extent of cytochrome f oxidation is about twice as largSc> in t,hcxmutant lwf.

THE

520.NM

CIL4NGE

IN

520nm

MUTANT

Absorpl~on

BAKLI~Y

Increase/

FIG. 1. The three recovery phases of the 520.nm absorption change aft,er Instrument time constants -lo-” set (Fig. lA, I)), and 3 X 10V5 set (Fig. lH, 1 mJ incident laser energy.

Eapid (R) recovery phase” (psec) Intermediate (I) phase6 (mscc) Slow (S) recovery phasec (msec) Cytochrome j osidationd (WC) Cytochrome f decal-? (msec) a Half-times h Cf. Figs. c Cf. Figs. d Cf. Figs. F Figs. 9B,

7 zt

3

0 +

4 zk 1.5 100 *

30

50 *

25

412

tracts

10 i 0.2

150 zt 50

5It3

measured from oscilloscope lB, I?, and 4R, R. lC, F, and K, F. 9A. C. 11.

2

f 2&l

2 0.1

a laser flash. C, E, and F’).

520

9+2 0.2

f

0.1

2fl

520 520

25 +

10

554

4 +

2.5

551

on t.ime scale

The S-phase spt~ctra, measured about 2.5 msec after the laser flash, are similar in shalw to the spectra in Fig. 3 (cf. l;ig. 11, Ref. 14). Since there are no recovery phases missing in the mutant barley leaf, as compared with the normal leaf, we may conclude that the major portion of the 520.nm absorption change is not dw to chlorophyll h. Low-temperature kimtics. At 100” I<, thta recovery kinetics arc similar for both thcl normal and mutant leaf (Fig. 4). The Rphaw recovery rate is slightly slower than thr room temprrntuw rate (I:ig. 4A and C,

of Figs.

lA4, I>, and 4A, 1).

IC - II/% = 10 ~wc, 100” I\). On the other hard, the I- and S-phase rates are faster than the room tompernture values. The Iphase 1~1~= 0.2 msec, a factor of 20 faster than at room temperature. The S-phase tl/a = 2 msec, :t factor of 30 faster than at room t,empcrature (Table II). The possibility that different procwses are involved at low temperature for the I and S phases was considered, and a temperature profile was t,alwn (Figs. 5 and 6). I:or both tht: normal (Fig. 5) and mutant leaf (Fig. ci), the R phwo is only slightly slower, below the

r,ow-tenl/w”alure

FIG. 2. Spectra of i ho rapid (I<) rr~~,very phase ill Ilormd arid mlltallt barley leaf. B I Fhaae (-100rsec

after loser floshi

spcti”a.

The

I?-

:111d

I-

phaw spectra at lo\\. tc~mpcraturr arc she\\-n for the normal (E’ig. 7) and mutant barley leaf (Fig. S). The peak wavelength is shift,ed from 520 to 525~.30 nm, possibly due t,o a relatively greater increase in scattering, by the frozen aamplrs, in the wgion br>lo\\-.520 nm. Cyloclrwntr! f Xirretics. The cytochromc J’ spectra (E’ig. 3) \vere measured from rccordings on the t’irne scale of E’ig. 9B and D. A distinct absorption decrease c:tll be obst!rwd, after the lawr flash, \vit’h a rccovrr> halftime of 4-S rns~‘c. (1~1 the faster time scalrs of Fig. 911 ad C, the signa-to-noise ratio is relatively lo\\; however, cJ%ochrome j” oxidation cm be mcasurcd with :I hnlft,ime of 50 pscc in the normal leaf arltl 2.5 pscc in the mutant ltxnf (Table II). This diffrrence in the cytochrome j’ kinetics may be due t’o the> lack of chlorophyll b in the mut,ant, resulting in II diffrwnt environment for cytochrome J’ at the trapping sit’cl. I~~rtir~clio~~

cocJfif:ietlts

ad

qua~rtuirr

requit,e-

nwrts. If the incident laser cncrgv is attenuutwl to givr light-lirnitcBt1 oxidation of I cvtochrome /‘, the curves of I’ig. 10 ~111 be c74mp .I I-~~~ obtained. From thtl initial slope, a quantum 4% 560 550 w rcquiwmrwt of 4 f 1 is calculated for both the normal and mutant’ leaf. The relativel! FIG. 3. Spectra of the itltermediate (I) phase, after a Q-switched laser flash, ill rlormal ant1 muhigh quantum rt~quirement~is partially due tant harl(:y leaf. to thrl fact tha,t a fraction of the lasrr light, at 094 run, is nbsorbcd by System II and is freezing point. Howw~r, the I phase shows not t+fective for c>%ochrome/’ oxidation. In a distinct break before the freezing point, the above calculation, an extinction coefgoing from 4-msec absorption increase at ficient of 16 rn31-1cm-‘, \vas used for cgto23” (Fig. 1B and E) to a 0.2.msrc partial chrome j at 554 nm (l(i). ;\Ieasurements of recovery at X0 (I*‘ig. 4H and E). AU inter- absolute absorption showed that 90 %Jof t,he mediate temperatures, the I phase occurs as incident laser light \vas absorbed by t’he a plateau, on the time scale of Fig. 4B and leaf samples. This value was used in the 12. above calculation, and cn11 be nscd to estiThe S phase gives the usual activation mate extinction coefficients Q , ~1, and ES energy curve between 23” and S” (Figs. 5 for the three phases at 520 nm as follows. and 6). In this experiment, the normal leaf I’rom light-limited curves similar to lcig. S phase t1i2 is 70 msec at 2X”, and 140 mwc 10, and assuming a quantum yield of unity at 8”. For the mutant leaf, 8 phase t1j2 is for the absorption change at 30 rim, the 120 mwc at 23”, and 160 msec at S”. Due results shown in Table III were obtained to the relatively large uncertainties involved (cf. Ref. 14). The [CYll]/P~,, values were in the calculation, no distinction can be made calculated using the AOD of the R, I, and S betel-een the activation energies for the phasesunder saturating light conditions. P‘or normal and mutant leaf (:3-7 kcal/mole). 111 the normal barley leaf [chl] Z 5 X lo-” the frozen state, the S phase shojvs orlly the mmole/cm2, and for th(x mutant leaf, [chl] N _ 2.5 X 1F mmol(bs,,cm”. Thra product,s: fast’er 2-mwc halftime (Figs. 5 and 6).

510nm Absorption

FIG. 4. The three recovery phases of the 5Wnm -lop6 set (Fig. 4A and D), -10e5 time constants -X and Fj. 1 mJ incident laser energy.

absorption WC

(E’ig.

change -LB

anti

at, 100” Ii. Instrumetlt and -1OV set (Fig.

I<:),

/‘RjyO X tn S 0.4 (normal leaf, units of Table III) and 0.5 (mutant leaf) cannot be comp:u~d with values in the literature due t,o the rapid time scale involved. However, for the rwr1n:r1 lexf, 1-‘,2, x E S 0.09 (1 and S ph:lscs) and for the mutant leaf, I-‘~zz~ X cI = 0.09 and /‘$,20 X ERZ 0.11. The Iattcar vducs can be compared \vith J’s20 X I!: G 0.1 reported by Witt :md Mtiller (17) for 10-4-sw fl:Lshw, and 1’;~” X 1~’ S 0.0s wlculntcd by Kok ei al. (IS) for spinach chlorophwts. .lhorptio~~ drmye at 520 ma. The main conclusion from the present work is that, chlorophyll 0 does not make a significant contribution to the absorption chungt xt 320 nm. Similar results have been obtained by Hill(lr (19), who sho\vtd that absorption ch:mge.sat 51.5 and MS nm were not corrclatcd, upon addition of fatty acids to Chlorclln. The altc~rnatr: hypothesis, that p-carotcw~ (5, 14, 20) is involved in the 520-nm absorption chxngr:, must, no~v bc considered. From Table 1, hhe WTimut:mt mtio for fi-c:troteikc~ is I .Ci. If one aver;qys the pc~k absorption ch:mgesfrom 12 spcctr:t, me obtains for the R phase, WT/mutnnt, = 1.2, :and for t,hc I and S’ phases, WT/ mutant8 = 1.7. The latter wlue correlates wit,11 the ratio for p-c:trot~cwr~in Table I. Hex-ever. the valw for thrt R phwr is more clorely rc~l:tted to the ratio for chlorophyll a in Table I, WT,‘mut:mt = 1.3. If chlorophyll (I tlow contribute to thrs R phase at 520 nm

,43 Temperature,

Ii5

1’

IGO

Cieqrees Kelv~r

Frc:. 5. The logarithm of the halftime for I hc t hrw phases at 520 nm in barley leaf vs i he reciprocal of the absolute temperature. ‘l’he ~~umbcrs itI parentheses indicate the number of mcas\lremonts averaged irrt 0 a single point

(?I), its effect must be rcl:ltivelv small, tluc: to the following two lines of ev~drnw. 1. Neither the pale-green mut:ult of Chlavr ydomonas (chl n t,o chl b ratio = :3) nor the blue-green alga, 1%m~?.idi7L?r~> sp. (only chl t\pc is chl a) showsany ph:rst: of the 5%0-nm absorption change (14). 2. Vrom Table I, [chl a + chl h]/[pcarotmlc] tquals 43, for normal barley, and 43 for mut:mt, barltsy. From Table III, t’hr:

Barley Mutant Leof (Def~uent In Chl b) 520nm

Barley

FIG. 8. Spectra ate (I) recovery barley leaf.

30

IO0

I 16) 1 i ‘14) j .& 300 250

260

I67

143

Temperature.Deqrees

FIG. 6. Halftime the mutant barley

FIG.

7. Spectra (I) recovery leaf.

III

100

Kelw

vs reciprocal temperature for leaf. See explanation for Fig. 5.

B0rkyLeaf

diate barley

-1%

WT) ‘7°K

of the rapid (R) RII~ illtermcphases at 77°K in t.he normal

a ratio [chl]/l’,?O = 40-50. If chlorophyll were the chief contributor to the R phaw, one would expect the latter ratio to be much lower. The fact that the ratio [chl],i Pjso is correlated with the [chl a + chl b]/ [fi-carotene] ratio is consistent with the proposal that the major portion of the initial 520.nm absorption change (R-phase) is due to fl- carotene. E’urther evidence for the involvement of carotenoids in the ZO-nm absorption change has been obtained by Mathis (22), who

Mutant Leaf, 77°K ichl b deftctenfl

of the rapid (R) and intermediphases at 77°K in the mutant

observed a similar absorption change in chlorophyll a-carotenoid-digitonin suspensions. The question remains as to what type of process causes the ,520.nm absorpt’ion change. If this change is associated with a light-induced change in membrane potential (3); e.g., a Stark effect’, then one would expect the absorption change to become stabilized at lo\v temperatures. The stabilizat’ion would be due to decreased electron conductivity and increased dielectric COIIstant of the membrane components at low tcmperaturc (23). However, Fig. 4 shows much faster recovery rates for the X0-nm absorption change, below the freezing point. Figures 5 and 6 indicate that different processes arp involved at low temperatures for th(k I and S phases. The rat’e for thr 1 before the phase undergoes a transition freezing point is reached; the S phase decay rate undergoes a transition across t.he freezing point. This sensitivity of thfl 1 and A’ phases to decreasing temperatuw may indicate changes in t’he state of the membraw in the vicinity of the freezing point,. Thus, the initial absorption increase at ZOrrm may bfl due to an int’craction of /%carotcnc: with an activated state of chlorophyll. The subsequent dark decay phases may then be dependent on the detailed state of the membrane. Analogous result,s exist for bacterial chromntophores, in which light-induced carotcwoid absorption changes have been studied (24). These carotcnoid changes can be induced by A%TI’ and pyrophosphatc (‘ls),

Loser

A

Laser

IB

Cyt t Oxidation 554nm Got )Kll

-

-1

-I

-IO

t 0

02

G3

iI4

65

ok

Gi

I-08

-1

09

IO

qcldentLaserEnergy(mj!

inhibited by uncouplers and :mtibiot,ics (26). The high quantum ykld of 3 for t,htb carot~woid changct indicates a structurxl chnllgr in thtb c~nvironmc~rtt of thn carotcnoid moleculw (Y), possibly a changc~ in tht, st:tt8c of t,ht) mr~mbrano. md

8

IIILI)I1I!xx

4. FORK, 1). C., AMESZ, ilnn. Rep. Curnegie (1965). 5. CHANCE,

R.,

1x1)

J., .\NU Iml. S.\GEI~,

32, 548 (1957). W. W., Plant

AIYI)ERSC)N, 12’ashingfon I:.,

J. M., 65,

/‘/un/

1X. KoI<, f’ell

ph!/sio/.

l!).

7. HIY~M.~, T., ~~‘ISHI.WJR.\, ibl., .\NI) (:II \N(‘E, B., 14naZ. Riochern. (to be puhlishcd). 8. FORK, D. C., ANI) AYESZ, J., .4nn. Rep. Cnrneyic 9 10. HIGHKIK, 11.

DEVAULY, FLING

H. It., I).,

66, Hiophys. Plant in “11

TECHNIQUES

160

(190(i). J. 9, A-120

phyniol. 26, \I’,,) &lISIN(: IN

ACAIJEMI[’

PRESS,

h’~w

YoltIc

(2. H. EDS.),

ANI)

(AY.P.)

(1969).

(IS. (:IUSON, p. 165.

A.,

I~ILLEJL,

%. Phys.

Chem.

I<. G.. Biwhiuz.

L.,

Y.\Nc,

I’lwnt

-4 eta 172, 546

Hiophys.

(1969). 20.

Fon~i,

21.

ion 6’7, 49G (19(S). ZElGEIZ, (:., RltiLLER,

1). C., iinn.

Hep.

(‘hemie

22. brATHIS,

I’.,

(‘arnegie

Inst.

A.,

.\NU

(.\-.F.)

29,

f’hofochem.

M’ushing-

WIW, H. T., z. 13 (1961).

Photobid.

9, 55 (1969).

B., KIH.II{.I, ‘I’., I)EVAULT, II., HILW., NIsHIMI~J~.~, Xl., l\~~ HIYAMA, T., in “Progress in Photosynthesis,” (H. Metsner, ed.) to hc published (1969). 21. Krwz, I. I)., i,o.wkr, P. A., .\XU CALWN, N., 23.

CKYNCE, IIJ~E’PH,

25.

R\LWXIEFFSKY,

fiicph!y.s.

(1W).

12. I)EV.IULT, Il., .ZNI) CH.IS(:E, I3., Bioph!/s. J. 6, 825 (19M). 13. RIACKINSEY, C;., J. Rid. Chem. 140,315 (1!)11). 14. HILUI~E’PH, W. W., Amos, M., ANI) CH.ZNCE, B., Plunt Phqsiol. 41, 983 (l!Mi). 15. HILDRE.I.H, W. W., Biochim. Hiophqs. .Irfa 153, 197 (1968).

~I~-LLER,

21, 1 (1959).

IS., C~OI’EII, B., .INI) Phy.sid 5, 373 (1963).

Physilictl.

294 (1950). .\SI) S.\Rl-

BIOUTEMISTRY”

CHANCE, 11. II. EISE.L.H.\KIW, ANI> I<. K. LOSJSERG-HOI.M,

ll.T.,

Leipziy

1’-‘Aysio/. 43, 303 (lN8).

6. HILDREW,

Inst. Washington HILDRETH, W.,

17. WJ~,

173

.I. 4, 227 M.,

(1964). :I rch.

Hiochem.

Hiophys.

130, G4G (1909). 2(i. ~LEIS~H.~~.\S,. I’hotochem. 2:.

AMESZ, JIENW

1). I<:., I’hofohiot.

ANJ) CL\yTON, 8, 287 (1968).

.I., AA-I) VJW)ESJJEIK;, IN PJI~,~~JSYN,~HESIS“

\NI) J. C. (;OEJ1HEEJt, ~:OW~:l~J) \,I (l!%(i).

EI%.),

w.

J(.

Ii.,

J., irk “CUE(J. B. TJIOM.\S 1’. 7%

I)oxKER,