Photoconversion and regeneration of active protochlorophyll(ide) in mutants defective in the regulation of chlorophyll synthesis

ARCHIVES

OF

BIOCHEMISTRY

Photoconversion in Mutants

AND

BIOPHYSICS

160, 43(t439

and Regeneration

Defective

of Active

in the Regulation OLE FREDERIK

Institute

(1974)

Protochlorophyll(ide)

of Chlorophyll

Synthesis’

NIELSEN

of Genetics, University of Copenhagen, @ster Farimagsgade

d A, DK -1363 Copenhangen

K.,

Denmark Received

August

31, 1973

Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-dl2 and tig-bz4/tig-b24 accumulate Z-fold, tig-o34/tig-o34 5- to g-fold, and lig-di2/tig-di2 15-fold more protochlorophyll(ide) than the wild type. The amount of photoconvertible protochlorophyll(ide) accumulated in darkness despite the large differences in total protois the same in all genotypes, chlorophyll(ide) content, indicating a constant number of photoconversion sites. When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and lo-min dark periods is higher in +/tig-d12, tig-bz4/ tig-b24, and tig-os’/tig-034, but lower in tig-d’z/tig-d12. There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll (ide) in leaves with the genotypes +/+, and tig-o34/tig-o34 are similar or identical. +/tig-03”,

Seedlings of etiolated angiosperms accumulate photoconvertible and some nonconvertible protochlorophyll(ide). Upon illumination the photoconvertible protochlorophyll(ide) is reduced to chlorophyll(ide) a. The photoconvertible portion of the protochlorophyll(ide) exists in a pigment-protein complex (23), which is extractable (12,22), called “protochlorophyll holochrome” (23, 24) and is bound to the membranes of the prolamellar body (10, 11). When &aminolevulinic acid is fed to etiolated leaves in darkness, protochlorophyll(ide) accumulates in much greater quantities than in untreated leaves (6). This shows that A-aminolevulinic acid synthesis limits the rate of chlorophyll syn1 This investigation was supported by grants from the National Institutes of Health, U.S. Public Health Service (GM 10819 to Professor D. von Wettstein); the Carlsberg Foundation; and the Danish Natural Science Research Council. 430 Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

Inc. reserved.

thesis (1, 7)) and regulation is thought to occur at the transcriptional level through the synthesis of S-aminolevulinic acid synthetase, which turns over rapidly and catalyzes the rate-limiting step in the pathway (15). However, the amount of protochlorophyll(ide) that is convertible to chlorophyll(ide) by a brief, but saturating, flash of light is not increased (7). Using weak red light, Sisler and Klein (21) showed that it was possible to produce more chlorophyll(ide) in &aminolevulinic acid-fed leaves than in control leaves. Sundqvist (26, 27) and Granick and Gassman (8) demonstrated that after a saturating light flash, with time in darkness, a portion of the inactive protochlorophyll(ide) becomes photoactive. Egneus and Sundqvist (3) obtained an action spectrum for the photoreduction of protochlorophyll(ide) synthesized in response to exogenous &aminolevulinic acid and found the maximal effectiveness of red light near 650 nm.

PHOTOCONVERSION

OF PROTOCHLOROPHYLL(IDE)

Evidence for the activation of inactive protochlorophyll(ide) in leaves that were not fed &aminolevulinic acid has been given by Granick and Gassman (8), and more recently by Virgin and French (30, 31). Several tigrina mutants in barley accumulate in darkness large amounts of protochlorophyll(ide) with an absorption maximum at 633 nm rather than at 650 nm (4, 16, 32). In this respect, these mutants resemble wild-type seedlings that have been fed d-aminolevulinic acid. The @ina mutants are considered to be constitutive mutants in regulat’ory genes for chlorophyll synthesis (4). More protochlorophyll(ide) in one of these mutants2 than in the wild type could be converted to chlorophyll(ide) by illumination with weak red light, and the 633-nm form of protochlorophyll(ide) dccreased during chlorophyll(ide) formation (16). Photoconversion of protochlorophyll(ide) in the mutant seemed to require more quanta per mole chlorophyll(ide) formed than in the wild type (4, 16). The present study shows that in etiolated seedling leaves of the regulatory mutants tiy-bz4, tipP, and tig-o34, the amount of protochlorophyll(ide) which can be converted with a saturating light flash does not exceed that in the wild type. The kinetics of the photoconversion of the initially convertible protochlorophyll(ide) in et.iolated leaves of seedlings lyith the genotypes +/+, +/tiy-034, and tiy-034/tiq-034 were indistinguishable. A portion of the initially inactive protochlorophyll(ide) absorbing maximally in the red region near 633 nm in the mutants could be activated to photoconvertible protochlorophyll(ide) with an absorption band near 650 nm. The parallel characteristics among the mutants examined here and &aminolevulinic acid-fed wild-type seedlings provide evidence that &aminolevulinic acid synthetase activity is regulated through the product,s of a number of nuclear genes. MATERIALS

AND

METHODS

Plant material. The plant material comprised wild-type barley, Hordeum vulgare L., cultivar Svalijf’s Bonus, and three tigrina mutants from Bonus: tig-b24, tig-d12, and tig-o3*. The mutant 2 lig-034, this named “infrared

mutant has provisionally 5” (33).

been

IN MUTANTS

431

fig-bz4 was obtained from a homozygous seedstock (33). The segregation of the other two mutants fitted the ratios: 1 wild type (+/+) to 2 +/tig@ to 1 tig-d12/tig-d12; and 1 wild type (+/tig-034) to 1 tig-034/tig-034, the latter being a balanced lethal stock (33). Seedlings were grown in darkness at 23°C in vermiculite and watered daily with tap water. On Day 7 the shoots of segregating stocks were detached, placed individually in small glass vials containing 1 ml of tap water, and stored in darkness at 4°C overnight. The seedlings were classified by removing the apical l-cm segments of the primary leaves and analyzing the segments for their greening capacity during an overnight period of illumination. Photoconversion of protochlorophyll(ide) to chZorophyZl(ide). The light source was a Braun electronic flashlight, model F 700 Professional. To obtain near-monochromatic light for the study of the kinetics of photoconversion of the initially photoconvertible protochlorophyll(ide), the light was filtered through a 640.nm Depil interference filter (no. 272442) and a red glass cut-off filter (RG 610), both from Jenaer Glaswerk, Schott und Gen., Mainz, Germany. For pigment extraction, segments of 25 primary leaves (tissue lying between 10 and 30 mm behind the apices) were irradiated with monochromatic light at a distance of 4 cm from the front of the flashlight, or with white light at a distance of 20 cm. Irradiations were performed at temperatures ranging from 6 to 10°C. For in vivo spectrophotometry the segments from six or seven primary leaves were cut into two and arranged in parallel to fill the glass window of a metal holder which could be fitted into the spe&ophotometer. White light irradiation of the samples at a distance of 20 cm from the flashlight and spectrophotometry were carried out at room temperature (20°C). A piece of paper tissue (Linella) was placed in the reference beam of the spectrophotometer to compensate the light scattering by the leaf pieces. Pigment determinations. Leaf segments were ground with a mortar and a pestle, with sand and an 857, (v/v) aqueous acetone solution containing 0.25% ammonia. Protochlorophyll(ide) and chlorophyll(ide) in the extracts were quant,itated as described earlier (17). Absorption spectra were recorded with a Cary 17 spectrophotometer equipped with a scattered transmission accessory and an EM1 9659 QB photomultiplier tube. RESULTS

Segments from lo-30 mm behind the of tho primary leaves of etiolated seedlings of wild-type barley contain be-

apices

432

OLE

FREDERIK

tween 13 and 16 nmoles of prot’ochlorophyll(ide) per g fresh weight (Tables I-III). Seedlings, carrying mutations in regulatory genes for protochlorophyll(ide) synthesis, accumulate protochlorophyll(ide) in amounts exceeding the wild-type level to extents depending on the gene involved and whether it is present in homozygous or heterozygous form. Segments of primary leaves of homozygous tig-d’2 seedlings contain 15 times more protochlorophyll(ide) than the wild type (Table I), while leaf segments of heterozygous tig-c-P2 seedlings accumulate approximately twice the amount found in the wild type (Table I). The mutant tig-bz4 also accumulates 2-fold more protochlorophyll(ide) than does the wild type (Table II). Seedling leaf segments of the

NIELSEN

mutant tig-034 contain five to six times as much protochlorophyll(ide) as the wild type (+/+ ), while leaf segments from seedlings heterozygous for tig-034 contain about the same amount of protochlorophyll(ide) as +/+ seedling leaf segments (Table III). The differing protochlorophyll(ide) contents of the leaf segments are reflected in the in vivo absorption properties. Absorption spectra of etiolated wild-type leaves (Fig. 1) and leaves of +/tig-034 have a maximum near 650 nm with a shoulder in the 635-nm region. Etiolated leaves of seedlings homozygous for tig-bz4 and those heterozygous for tig-d12 (Fig. 2) have absorption spectra which are qualitatively identical: The main absorption maximum in the red region due to protochlorophyll(ide) is near 635 nm with a

TABLE

I

CONTENT OF GREEN PIGMENT, PHOTOCONVERSION OF ACTIVE PROTOCHLOROPHYLL(IDE) PRESENT INITIALLY, AND REGENERATION OF PHOTOCONVERTIBLE PROTOCHLOROPHYLL(IDE) DURING DARK PERIODS AFTER A PAIR OF LIGHT FLASHES 1. COMPARISON OF +/+, +/tig-cl’2 AND tig-cP/tig-d12 LEAF SEGMENTS. Genotype

Treatment

+ &d’2

+/+ Green pigment (nmoles/g fresh wt)

Darkc Two flashes Four flashes Two flashes + 4 min darkness + two flashes Two flashes + 10 min darkness + two flashes Average pigment content and standard deviation

Totale

Chlorophyll(ide)

15.8 13.3 22.4d 14.6 16.1 14.8 15.0 14.7

0.8 0.6 10.4 8.8 10.2 9.2 12.1 11.3

15.0 14.5

12.7 12.2

14.9 f

0.8

Photoconversion (%P

Green igment (nmolesfg fresh wt) ~

Green pigment (nmoles/g fresh wt)

Totala

Chlorophyll(ide)

60 63 62 81 77

36.8 24.1 27.1 24.5 32.6 22.2 25.7 24.8

0.6 0.5 10.7 9.6 10.1 9.6 13.7 12.8

209 234 164 225 234 225 232 234

0.6 0.7 9.3 9.0 9.8 9.2 11.4 10.7

85 84

28.2 23.6

16.4 14.1

229 244

12.2 11.6

5 5

27.0 f

4.5

a Sum of protochlorophyll (ide) and chlorophyll (ide). b Percent protochlorophyll(ide) plus chlorophyll(ide) found as chlorophyll(ide). c Chlorophyll(ide) found is due to photoconversion elicited by the green safelight for vision. d Not included in average pigment content, because the aberrantly high total ascribed to errors in sorting the seedings.

Totala

223 f

Chlorophyll(ide)

23

used when required pigment

content

is

PHOTOCONVERSION

OF PHOTOCHLOROPHYLL(IDE) TABLE

OF GREEN

CONTENT INITIALLY,

AND

433

II

OF ACTIVE PROTOCHLOROPHYLL(IDE) PRESENT OF PHOTOCONVERTIBLE PROTOCHLOROPHYLL(IDE) DURING 11-4~~ PERIODS AFTER A PAIR OF LIGHT FLUSHES 2. COMP.IRIS~N OF +/+ (BONUS) and tig-b24/tig-b24 LE.~F SEGMENTS PIGMENT,

PHOTOCONVERSION

REGENERATION

Genotype

Treatment + /+

Totala

Darkc Two flashes Four flashes flashes + 4 min darkness + 2 flashes Two flashes + 10 min darkness + 2 flashes Two minutes continuous white light Average pigment content and standard deviation

13.5 14.5 14.6 14.1 14.9 14.5 12.9 14.3 13.6 14.2 13.9 13.5 14.0 + 0.6

tig-P4/tig-bz4

(Bonus)

Green pigment (nmoles/g fresh wt)

Two

IN MUTANTS

Chlorophyll(ide) 0.3 0.4 9.9 9.6 10.0 10.1 10.2 11.6 11.6 12.1 10.4 10.5

Photoconversion (%I* 2 3 68 68 67 70 79 81 85 85 75 78

Green pigment (nmoles/g fresh wt) Total”

Chlorophyll(ide)

27.6 28.2 27.7 27.4 29.0 27.8 26.1 26.4 27.4 27.8

27.5 f

0.6 0.9 10.1 10.7 11.5 10.8 14.1 14.3 15.6 15.8

2 3 37 39 40 39 54 54 57 57

0.8

n Sum of protochlorophyll(ide) and chlorophyll(ide). b Percent of protochlorophyll(ide) plus chlorophyll(ide) found as chIorophyll(ide). c ChlorophylI(ide) found is due to photoconversion elicited by the green safelight for vision.

shoulder around 650 nm. The absorption spectra of etiolated leaves of seedlings homozygous for tig-c112 (Fig. 3) and those homozygous for tig-034 (Fig. 4) appear to contain a single protochlorophyll(ide) absorption band with a peak at 633 nm. A brief, but saturat,ing, light exposure of et’iolated seedling leaves leads to the reduction of the photoconvertiblc protochlorophyll(ide) present initially. In this study a light treatment of two flashes, 3-4 set apart was sufficient to saturate the photoconversion (cf. Tables I-III). The absorption of newly formed chlorophyll(ide) in vivo has also been compared among mutant and wild-type seedlings. Although rapid early shifts in absorpt’ion (2, 5, 20) were progressing before the spectral scans began, the differences among the various spectra are meaningful, because the irradiations and spectroscopy were per-

Photoconversion (%YJ

used when required

formed in a uniform manner. The absorption wavelength of the absorption maximum in vivo of the initial chlorophyll(ide) varies with the genot’ype and thus with the protochlorophyll(ide) content of the seedlings. In spect’ra of +/+ (Fig. 1) and +,‘tig-034 seedling leaves, the absorption maximum is near 682 nm with a weak shoulder on the blue side of the peak. Spectra of tig-bz4/tig-bz4, and +/tig-d12 seedling leaves show an absorpt’ion peak (cf. Fig. 2) near 675 nm with a shoulder on the red side of the peak. Seedling leaves which are homozygous for tig-cl12 and tig-03*, respectively, have chlorophyll(ide) absorption spectra with a maximum near 675 nm (Figs. 3 and 4), as determined from difference spectra. During a subsequent dark period, the chlorophyll(ide) absorption maximum in wild-type (Fig. 1) and +/tig-03* leaves shifts toward the blue from 682 nm to

434

OLE

FREDERIK TABLE

NIELSEN III

CONTENT OF GREEN PIGMENT, PHOTOCONVERSIONOF AGTIVE PROTOCHLOROPHYLI~(IDE)PRESENT INITIALLY, AND REGENERATION OF PHOTOCONVERTIBLEPROTOCHLOROPHYLL(IDE)DURING DARK PERIODSAFTER A PMR OF LIGHT FLASHES 3. COMPARISONOF +/+ (BONUS), +/tig-oa4, AND tig-o”/tig-034 LEAF SEGMENTS Genotype

Treatment + /+

Green pigment (nmoles/g fresh wt) Total”

Two flashes Four flashes Two flashes + 10 min darkness + two flashes Average pigment content and standard deviation

Chlorophyll(ide) 11.3 11.5 10.2 11.2 11.6 12.4

14.4 14.6 13.1 14.2 13.3 14.4

14.0 f

+ /tig-o*

(Bonus) Photoconversion (%T

78 79 78 79 87 86

0.6

tig-o”/tig-0”

Green pigment (nmoles/g fresh wt)

Green pigment (mnoles/g fresh wt)

Totala

Totala

Chlorophyll(ide) 9.9 11.3 11.5 10.7 12.9 12.9

27.7” 22.4” 19.7 14.5 14.8 17.5

16.6 f

2.5

a Sum of protochlorophyll(ide) and chlorophyll(ide). b Percent of protochlorophyll(ide) plus chlorophyll(ide) found as chlorophyll(ide). c Not included in average pigment content, because the aberrantly high pigment to errors in sorting the seedlings.

672 nm, i.e., the Shibata-shift occurs (19). A blue shift of the chlorophyll(ide) absorption peak to 672 nm occurs also in homosygous tig-bz4, heterozygous tig-cP2 (Fig. 2), homozygous tig-d12, and homozygous tig-034. Photoconversion results in a decrease of the protochlorophyll(ide) absorption in the 625- to 655-nm region. After the initial conversion with flashed light, the absorption spectra retain a band at 630-635 nm with a shoulder near 650 nm (Figs. 1 and 2). This shoulder is not due to photoconvertible protochlorophyll(ide) that was present before the flashes, because as noted previously, the flashes are saturating, and because it has been shown that the 650-nm shoulder can be found also after a 4-fold increase of the amount of light given. During a prolonged period in darkness, the height of the 650-nm. shoulder increases with a simultaneous decrease in absorption at 633 nm (Figs. 1 and 2). If, thereafter, a second pair of light flashes is given, the chlorophyll(ide) absorption increases, while protochloro-

Chlorophyll(ide)

66.6 76.8 80.2 84.9 76.9 68.9

75.7 f

content

11.0 10.8 11.7 11.6 14.6 14.4

6.9

is ascribed

phyll(ide) absorption drops (Figs. 1, 2, 3). It is concluded that the increase of 650-nm absorption during the dark period reflects the transformation of inactive protochlorophyll(ide) absorbing at 633 11111to photoactive protochlorophyll(ide) absorbing principally at 650 nm. The transformation of 633-nm protochlorophyll(ide) into chlorophyll(ide) has been followed with repeated light flashes over a 30min period in the mutant tig-034 (Fig. 4). After an initial spectral scan, the leaf sample received a pair of white light flashes to convert all the active protochlorophyll(ide) present in darkness. The sample was then exposed to successive single-flash treatments given at intervals of loo-120 sec. Absorption spectra were recorded after each flash treatment (Fig. 4). All spectra except the dark spectrum form two isosbestic points, one near 648 and one close to 622 nm. This result is in agreement with the notion that the first light treatment reduces to chlorophyll(ide) the protochloro-

PHOTOCONVERSION

OF PROTOCHLOROPHYLL(IDE)

435

IN MUTANTS

J FIG. 1. Absorption spectra of a representative sample of etiolated wild-type leaves; (--) dark; (-----) rescan within 1 min after exposure to a pair of flashes of white light; (. . . . .) rescan after a dark period of 8 min after the flashes; (-*-.-) rescan after a dark period of 10 min and a second pair of light flashes.

FIG. 3. Absorption sample] of etiolated Fig. 1.

0.1

600

spectra of a representative tig-d12/tig-d12 leaves, as in

610

670

690

710

0.4

0.1

FIG. 2. Absorption spectra of a representative sample of etiolated +/tig-dlz leaves, as in Fig. 1.

phyll(ide) with a 650-nm absorption maximum which is present initially, while each following light burst converts quantit’atively into chlorophyll(ide) a portion of the protochlorophyllfide) absorbing initially with a maximum at 633 nm and which has been activated during the intervening dark period. Regeneration of active protochlorophyll(ide) has been quantitated by analyzing the pigments in acetone extracts of leaf samples. In order to minimize de novo synthesis of active protochlorophyll(ide), the experiments were execut,ed at 6-10°C. The close agreement of the values for chlorophyll(idc) and protochlorophyll(ide)

0.0 WAVELENGTH

(nm)

FIG. 4. Absorption spectra of a representative sample of etiolated tig-o34/tig-o34 leaves showing the formation of chlorophyll(ide) from initially active protochlorophyll(ide) and from subsequently activated protochlorophyll (ide). See text for details.

in duplicate samples of the wild type (Tables I-III)

is taken

as evidence

for

little

varia-

tion among the segments included within the samples. Furthermore, the small variation (standard deviation) in the total pigment content-14.3 f 0.8 (n = 27) nmoles per g fresh weight-indicates t’hat the extraction procedure is reproducible.

436

OLE

FREDERIK

In the wild type, a constant total pigment content is observed throughout the various treatments covering periods up to 10 min (Tables I-III), indicating that synthesis de novo of protochlorophyll(ide) does not occur during this period. The amounts of chlorophyll(ide) extracted after a series of two or four flashes of light, respectively, are approximately equal (Tables I-III). It follows that the conversion of the initially active protochlorophyll(ide) is saturated with two flashes of light, and regeneration of photoconvertible protochlorophyll(ide) during the time required to give t’he third and fourth flashes can be ignored. In the wild type the initial amounts of photoconvertible protochlorophyll(ide), determined as the averages of t’he chlorophyll(ide) contents of two and four flash treatments combined in Tables I-III without correcting for the minor amounts of chlorophyll(ide) present in the dark (cf. Tables I and II), are 9.7,9.9, and 11.1 nmoles per g fresh weight, representing 62, 68, and 79% of the total pigment content, respectively. The amount of initially photoconvertible protochlorophyll(ide) per g fresh weight in leaf segments of seedlings heterozygous or homozygous for one of the mutant genes are not significantly different from the wild type: 10.0 nmoles in +/tig-d12 and 9.3 nmoles in tig-d12/tig-d12 (Table I), 10.8 nmoles in tig-b24/tig-b24 (Table II), 10.9 nmoles in +/tig-034 and 11.4 nmoles per g fresh weight in tig-034/tig-034 (Table III). With dark periods of 4 or 10 min inter-

NIELSEN

vening between the first and the second pairs of flashes, the amount of chlorophyll(ide) formed is larger than with no experimental interval of darkness separating the pairs of flashes because of the regeneration of active protochlorophyll(ide) during the dark period. This is true for all of the genotypes st’udied (Tables I-III). The regeneration occurring during the experimental dark periods inserted between the pairs of flashes can also be noted in the wild type and tig-b24 by the increase in chlorophyll(ide) as percent of total green pigment (Tables I-III). The mean rates of regeneration during 4and lo-min dark periods (Table IV) have been calculated from the differences in the means of the chlorophyll(ide) contents of samples of leaf segments given two pairs of flashes with and without dark periods intervening between them. The regeneration in +/tig@ during 4 min is 1.7 times, and during 10 min 1.9 times higher than in the wild type. Regenerations are 3.6 and 2.6 times higher in tig-b24/tig-b24 than in the wild type, whereas in tig-oz4/ tig-034 the regeneration during a lo-mm dark period is 2.2 times that in the wild type. In the heterozygote +/tig-034 1.4 times as much active protochlorophyll(ide) is regenerated during a lo-min dark period as in the wild type. On the other hand, regeneration is slow in tig-d12/tig-d12, occurring at only three fourths the rate found in the wild type. Thus, the presence of moderately greater amounts of inactive protochlorophyll(ide)

TABLE REGENERATION

OF PHOTOCONVERTIBLE PERIODS INTERVENING

Genotype

I-/+ -+/tig-d12 tig-dr2/tig-dr2

+/+

(Bonus)

tig-b24/tig-b24 +/+ (Bonus) +/tig-oaa tig-oa4/tig-os4

IV

PROTOCHLOROPHYLL(IDE) DURING 4BETWEEN PAIRS OF LIGHT FLASHES

Rate of regeneration of active protochlorophyll(ide) (nmoles. (g fresh wt)-r. mini)

AND

10.MIN

DARK

Regeneration in percent of initially active protochlorophyll(ide)

During 4 min

During 10 min

During 4 min

During 10 min

0.50 0.85 0.36 0.21 0.75

0.28 0.54 0.23 0.18 0.46 0.13 0.18 0.29

21 35 15 8 27

29 55 24 18 41 12 16 24

PHOTOCONVERSION

OF PROTOCHLOROPHYLL(IDE)

than in the wild type, as in the cases of tig4P4/tig-bz4, +/tig-d12, and tig-034/tig-034 leaf segments, is associated with increased regeneration of photoconvert’ible protochlorophyll(ide) per unit time. Extensive accumulation of inactive protochlorophyll(ide), as in tig-d12/tig-d12, is associated with a decrease in the regeneration rate below that of the wild type. The regcnerat,ion is nonlinear with t’ime, as the regeneration during IO-min dark periods is less than 2.5fold higher than during 4min periods. A possible way of determining if the photoconversion sites of protochlorophyll(ide) holochrome in the tigrina mutants are altered is to compare the kinetics of the photoconversion of the initially photoconvertible protochlorophyll(ide) in the mutants with the kinetics in the wild type. To minimize the regeneration of active protochlorophyll(ide) during the experimental irradiations, a series of varying numbers of monochromatic light flashes, rather than continuous weak light, has been used to obtain the kinetics. The fractions of active protochlorophyll(ide) remaining in segments of leaves with the genotypes +/+, +/tig-034, and tig-034/tig-034 after various doses of light, ranging from one through nine flashes, have been determined (Fig. 5). The regeneration of active pigment in leaves of the three genotypes during the period of flashing was found to be negligible. The progress of photoconversion in the three genotypes is similar if not identical, and is consistent with the earlier observations (17), that the photoconversion of protochlorophyll(ide) in barley leaves follows neither first- nor second-order kinetics. It may be pointed out that regeneration of active protochlorophyll(ide) might have taken place in earlier kinetics studies (17 and literature cited there) as is indicated by comparing the results of 2-min illuminations with flash treatments in Table II. In the present work, this source of error has been minimized. The activation of initially inactive protochlorophyll(ide) while photoconversion progresses in weak light would cause the kinetic analysis to approach first-order kinetics more closely t’han in the abspnnceof activation.

IN MUTANTS

FIG.

5. Semilogarithmic plot of the progress of photoconversion of protochlorophyll(ide) in etiolated leaves, using 640.nm flash light: 0 +/$; V +/tig-034; 4 tig-os4/tig-034.Each point represents the pigment analysis of the extract from a single sample of leaves.

It is concluded t’hat mutation of a regulatory gene resulting in constitutive protochlorophyll(ide) synthesis does not interfere with the formation of or the function of initially photoactive protochlorophyll(ide) holochrome. DISCUSSION

When segments from between 10 and 30 mm from the apex of primary leaves of wildtype barley seedlings are illuminated with a short, but saturating, pulse of light, between 60 and 79% of the total protochlorophyll(ide) is reduced to chlorophyll(ide). This corresponds to values between 8.8 and 11.5 nmoles protochlorophyll(ide) per g fresh weight. Brodersen (unpublished results) found a total protochlorophyll(ide) content per etiopla,st of barley seedling leaves of 7.1OV nmoles. Subtracting the inactive protochlorophyll(ide) and assuming that each conversion site contains one active protochlorophyll(ide) molecule, the number of sites per etioplast would be between lo6 and 107. Tigrilha mutants, mutated in regulatory

438

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FREDERIK

genes of protochlorophyll(ide) synthesis, accumulate between 2 and 15 times more protochlorophyll(ide) than the wild type, thus resembling wild-type leaves fed 6aminolevulinic acid. In spite of the accumulation of protochlorophyll(ide), the mutants contain the same initial amount of photoconvertible protochlorophyll(ide) as the wild type and thus the same number of conversion sites (cf. Tables I-III). Nevertheless, the rate of regeneration of active protochlorophyll(ide) varies with the protochlorophyll(ide) content: the rate is greater when a moderate amount of protochlorophyll(ide) is accumulated, but is smaller with extensive accumulation. The same results have been found previously in comparing wild-type wheat leaves with and without prior feeding with &aminolevulinic acid (28). The photoconversion of all the initially convertible protochlorophyll(ide) molecules in wild-type leaves by a brief illumination leads to chlorophyll(ide) with an absorption maximum at 678 nm, which within seconds is transformed into chlorophyll(ide) absorbing at 684 nm (2, 5, 20). This in viva chlorophyll(ide) form is then shifted to a 672-nm form (19). A 675-nm form of chlorophyll(ide) as primary photoproduct can be obtained in two ways, either as the result of a mixture of chlorophyll(ide) molecules in the conversion sites with large amounts of inactive protochlorophyll(ide) (cf. this study, 27) or as the result of a mixture of chlorophyll(ide) molecules in the conversion sites with photoactive protochlorophyll(ide) molecules at low photoconversion degrees in wildtype leaves (cf. 13, 14). The favored explanation for the 675-nm form of chlorophyll(ide) is that it results from interaction with either photoactive or photoinactive protochlorophyll(ide). It may well be that this interaction prevents the red shift to 684 nm, which is observed after complete photoconversion in the wild type. Nikolaeva et al. (18) found that NADPH could stimulate activation of 634-nm protochlorophyll(ide) after illumination of a homogenate of etiolated corn seedling leaves. Likewise, Horton and Leech (9) demonstrated that protochlorophyll(ide) absorbing at 630 nm in isolated etioplasts can be re-

NIELSEN

activated through exposure to a treatment with alternating light and dark periods in the presence of ATP. However, the activation of 630-nm protochlorophyll(ide) could not be pursued beyond the level of initially active protochlorophyll(ide) in freshly isolated etioplast preparations. Therefore, the authors suggested that there is always a pool of 630-nm protochlorophyll(ide) which cannot be activated. Also in plants fed &aminolevulinic acid (28) and in the mutant tig-tY4 (cf. Fig. 4) the regeneration has ceased before the pool of inactive protochlorophyll(ide) was exhausted. Further experiments are required to study whether there exist two forms of inactive protochlorophyll(ide). Earlier results have been interpreted to mean that de novo synthesis of protochlorophyll(ide) can take place in the light in plants containing large amounts of d-aminolevulinic acid-induced, inactive protochlorophyll(ide) (7). The demonstrations of the generation of active protochlorophyll(ide) from the inactive form in d-aminolevulinic acid-fed seedlings and in seedlings with genetic defects in the repression of the porphyrin pathway necessitates a new analysis of the problem of the derepression of the pathway. The question remains whether de novo synthesis of protochlorophyll(ide) can proceed before all t’he inactive protochlorophyll(ide) has been eliminated by activation or by bleaching. ACKNOWLEDGMENTS I thank Professors D. von Wettstein and A. Kahn for their advice and useful discussions. The skillful technical assistance of Mrs. Betty Netting, Miss Karen Birgit Pauli, and Mr. Poul Eriksen is gratefully acknowledged. REFERENCES 1. BOGORAD, L., LABER, L., AND GASSMAN, M. (1968) in Comparative Biochemistry and Biophysics of Photosynthesis (Shibata, K., Takamaya, A., Jagendorf, A. T., and Fuller, R. C., eds.), pp. 299312, University of Tokyo Press, Tokyo. 2. BONNER, B. A. (1969) Plant Physiol. 44, 739747. 3. EGNI~US, H., AND SUNDQVIST, C. (1970) Photosynthetica 4, 81-83. 4. FOSTER, R. J., GIBBONS, G. C., GOUGH, S.,

PHOTOCONVERSION

OF PROTOCHLOROPHYLL(IDE)

HENNINGSEN, K. W., KAHN, A., NIELSEN, 0. F., AND VON WETTSTEIN, D. (1971) in Proc. First. Eur. Biophys. Congr. (Broda, E., Locker, A., and Springer-Lederer, H., eds.), Vol. IV, pp. 137-149, Verlag der Wiener Medizinischen Akademie. 5. GSSSMAN, M., GRANICK, S., AND MAUZERALL, D. (1968) Biochem. Biophys. Res. Commun. 32, 295300. 6. GRANICK, S. (1959) Plant. Physiol. 34, Suppl. ..* xvm.

7. GRSNICK, S. (1963) in Proc. Fifth Int. Congr. Biochem. Moscow 1961, Vol 26 (VI) (Tamiya, H., ed.) pp. 176-186, PWNPolish Scientific Publishers. 8. GRANICK, S., AND GASSMAN, M. (1970) Plant Physiol. 46, 201-205. 9. HORTON, P., AND LEECH, R. M. (1972) Fed. Eur. Biochem. Sot. Lett. 26, 277-280. 10. KAHN, A. (1968) Plant Physiol. 43, 1769-1780. 11. KAHN, A., THORNE, S. W., AND BOARDMAN, N. K. (1970) J. Mol. Biol. 48, 85-101. 12. KRASNOVSKII, A. A., AND KOSOBUTSKAYA, L. M. (1952) Doklady Akad. Nauk. SSSR 86, 177. 13. LITVIN, F. F., AND BELYAEVA, 0. B. (1971) Photosynthetica 6, 200-209. 14. M~THIS, P., AND SAUER, K. (1973) Plant Physiol. 61, 115-119. 15. NADLER, K., AND GRANICK, S. (1970) Plant Physiol. 46, 240-246. 16. NIELSEN, 0. F. (1970) in Microscopic Electronique 1970. Comm. Sept. Congr. Int. Grenoble (Favard, P., ed.), Vol. III, pp. 179-180, Sot. Franc. Microsc. Electr. Paris. 17. NIELSEN, 0. F., AND KAHN, A. (1973) Biochim. Biophys. Acta 292, 117-129. 18. NIKOLAEVA, L. F., R~SKIN, V. I., AND ZAKIROV, A. (1972) Biol. Nauki 16, 70-74.

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19. SHIBATA, K. (1957) J. Biochem. 44, 147-173. 20. SIRONVAL, C., BROUERS, M., MICHEL, J.-M., AND KUIPER, Y. (1968) Photosynthetica 2, 268-287. 21. SISLER, E. C., AND KLEIN, W. H. (1963) Physiol. Plant. 16, 315-322. 22. SMITH, J. H. C. (1952) Carnegie Inst. Washington Yearb. 61, 150-153. 23. SMITH, J. H. C., AND BENITEZ, A. (1954) Plant Physiol. 29, 136-143. 24. SMITH, J. H. C., AND KUPKE, D. W. (1956) JVature (London) 178, 751-752. 25. SMITH, J. H. C., AND YOUNG, V. M. K. (1956) in Radiation Biology (Hollaender, A., ed.), Vol. III, pp. 343391, McGraw-Hill, New York. 26. SUNDQVIST, C. (1969) Physiol. Plant. 22, 147156. 27. SUNDQVIST, C. (1970) Physiol. Plant. 23, 412422. 28. SUNDQVIST, C. (1973) Physiol. Plant. 28, 464470. 29. THORNE, S. W. (1971) Biochim. Biophys. Acta 226, 113-127. 30. VIRGIN, H. I., AND FRENCH, C. S. (1972) in Carnegie Inst. Washington Yearb. 71, 187198. 31. VIRGIN, H. I., AND FRENCH, C. S. (1973) Physiol. Plant. 20, 350-357. 32. VON WETTSTEIN, D., HENNINGTON, K. W., BOYNTON, J. E., KANNANGARa, G. C., AND NIELSEN, 0. F. (1971) in Antonomy and Biogenesis of Mitochondria and Chloroplasts (Boardman, N. K., Linnane, A., and Smillie, R. M., eds.), pp. 205-223, NorthHolland, Amsterdam. 33. VON WETTSTEIN, D., AND KRISTIANSEN, K. (1973) Barley Genet. Newslett. 3, 113-117.