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Enhanced chlorophyllide accumulation after flash irradiation of etiolated wheat plants treated with SAN-9789
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• ••••• AL.F. © 1997 by Gustav Fischer Verlag, Jena
Enhanced Chlorophyllide Accumulation after Flash Irradiation of Etiolated Wheat Plants Treated with SAN-9789 GAUNA T. ]AHOUBJAN
and
IVAN
N.
MINKOV
University of Plovdiv, Plant Physiology Department, Tsar Assen 24 St., 4000 Plovdiv, Bulgaria Received February 24, 1997 . Accepted June 18, 1997
Summary
The accumulation of Chlide was investigated in flash-irradiated dark-grown plants as dependent on the decreased carotenoid content. Wheat plants (Triticum aestivum L.) were treated with two concentrations (10 and 100 ~mol/L) of norflurazon (SAN-9789) during germination, which caused inhibition of carotenoid synthesis. When SAN-treated plants were grown in darkness the amount of aCCJ,lmulated protochlorophyllide increased. After an irradiation of carotenoid-deficient etiolated plants with an increasing number of flashes (1-6) the decrease of protochlorophyllide and the accumulation of chlorophyllide were faster in comparison with the non-treated ones. The flash irradiation of carotenoid-deficient plants caused phototransformation of a greater number of originally accumulated protochlorophyllide molecules to chlorophyllide, and in that way the ChI synthesis that had been stopped in darkness could continue more efficiently.
Kry words: Wheat, Triticum aestivum L., protochlorophyllide, chlorophyllide, SAN-9789 (norjlurazon), flash irradiation. Abbreviations: Chl = chlorophyll; Chlide = chlorophyllide; Pchlide = protochlorophyllide; SAN-9789 = norflurazon; PLBs =prolamellar bodies; PTs = prothylakoids. Introduction
In dark-grown angiosperms the proplastids develop into etioplasts instead of chloroplasts, and protochlorophyllide (Pchlide) accumulates as a main plastid pigment (Virgin et al., 1963, 1981). Pchlide-protein complexes characterised by their fluorescence and absorption spectra are present in at least three spectral forms in inner etioplast membranes: Pchlid~50_657 and Pchlid~36-657' localised mainly in the prolamellar bodies (PLBs) (Ryberg and Sundqvist, 1982 a, b; Ikeuchi and Murakami, 1983; Lindsten et al., 1988), and Pchlid~28_633' localised mainly in the prothylakoids (PTs) (Lindsten et al., 1988). The irradiation of etiolated plants induces a transformation of Pchlide650-657 and Pchlid~36-657 to chlorophyllide (Chlide), whereas Pchlid~28-633 is considered a non-phototransformable form (Kahn et al., 1970). The photoconversion of Pchlide to Chlide involves a formation of j. Plant PhysioL Wll. 151. pp. 649-653 (1997)
several Chlide forms (Litvin et al., 1976, 1981; Dujardin, 1978, 1990). The treatment of plants with norflurazon (SAN-9789) causes an inhibition of the carotenoid synthesis, and the precursors phytoene and phytofluene accumulate (Bartels and McCullough, 1972). The effect of SAN-9789 on the plastids could be connected to the photoprotective role of carotenoids for chlorophyllide sensitised photodestruction (Krinsky, 1968; Ryberg et al., 1981) or to their structural role (Axelsson et al., 1981, 1982; Dahlin et al., 1983). When plants are grown in darkness, the total content of Pchlide is increased in SAN-treated plants. There is no structural difference in the PLBs between non-treated and treated plants (Klockare et al., 1981). This tendency is kept immediately after the phototransformation of Pchlide (1 s, week red light). During the following dark period (1 to 3 h) the photodecomposition of Chlide and the structural changes in PLBs
650
GAUNA T. JAHOUBJAN and IVAN N. MINKOW
occur faster in the treated leaves than in carotenoid-containing ones (Klockare et al., 1981; Ryberg et al., 1981). When carotenoid-deficient plants develop in light, the early lightinducible proteins (ELIPs) are specifically induced (Adamska et al., 1992). According to Axellson et al. (1981, 1982), the Chi content in plants grown under continuous red light at higher SAN9789 concentration is lower and is due to the inhibition of Chi synthesis and the faster decomposition of the Chi formed. On the other hand, we have previously shown (Minkov and Jahoubjan, 1993) that the total Chi content was higher during the first hour of irradiation (weak red or white light) of dark-grown SAN-treated plants compared with nontreated ones, which was followed by a decrease of Chi, most probably due to a progressing photodecomposition. The reason for the increased Chi content in greening carotenoiddeficient leaves is not known. In this paper we investigated the accumulation of ChIide in flash-irradiated dark-grown plants treated with SAN-9789 in dependence of the decreased carotenoid content, as well as the increased accumulation and re-accumulation ofPchIide in darkness. Material and Methods
Plant mauritz! Seeds of wheat (Triticum aestivum L., cv. Pobeda) were soaked for 24 h in water, or in water containing SAN-9789 (Sandoz LTD, Switzerland) in two different concentrations, i.e. 10 and 100 ~oll L. The seedlings were grown in darkness at 25 ·C for 8 days. The Pchlide in the dark-grown leaves was phototransformed to Chlide by a different number of flashes (1 ens of «white light» followed by 5 s of darkness, impulse energy 120 J) at a distance of 20 em.
Pigment analysis The contents of Pchlide, Chlide and carotenoids were determined in 85 % acetone. The extracts were measured fluorometricaIly on an LS-3B Perkin-Elmer spectrofluorometer and their emission spectra (600-750 nm) were recorded at an excitation wavelength of 440 nm. The pigment content was calculated using the molar extinction coefficients of Kahn (1983) for Pchlide and of MacKinney (1941) for Chlide. The phototransformation of Pchlide to Chlide is presented as a ratio Chlide/Pchlide, calculated on the basis of their fluorescence maxima in acetone extracts. The carotenoid content was determined according to MacKinney (1941). The mean values were based on 12 repetitions with 10 plants in each one. The mean deviations (SE) were calculated according to Student and only the values with p<0.05 were discussed. The low-temperature emission spectra (600-750 nm; excitation wavelength 440 nm) of the detached leaves were recorded at 77 K before and after flash irradiation. The spectra were normalised at the same value at 633 nm. On the basis of these spectra the ratios PchlidC(;57/PchlidC(;33 and ChlidC(;95/PchlidC(;57+ PchlidC(;33 were calculated.
220 ~ g-1 &esh weight in non-treated leaves, whereas it was 93 and 34/J.gg-1 at treatment with 10 and 100/J.mo1lL SAN9789, respectively. The SAN-treatment had no effect on the water content and the dry weight of etiolated and briefly irradiated plants. The latter varied about 11 % in non-treated and treated plants. After irradiation of the dark-grown plants with a different number of flashes, higher amounts of ChIide accumulated in the carotenoid-deficient plants compared with the non-treated plants. In the course of flash irradiation (1 to 6) the ChIide content increased from 9.6 to 18.1 /J.gg-1 fresh weight in non-treated plants, from 12.9 to 25.8 /J.g g-l at 10/J.mo1lL SAN and &om 17.1 to 29.7/J.gg-1 at 100/J.mo1lL SAN (Fig. 1). After irradiation of the dark-grown plants with 6 flashes, the Pchlide content decreased 3.4, 4.0 and 5.2 times in non-treated, 10 and 100/J.M SAN-treated plants, respectively {Fig. 2).
-
•
non·treated plante
~ 10 Ilmoi/L SAN·9789
a 30
o 100 Ilmoi/L SAN·9789
'i it .c
., • 20
.t:
0;-
at at
::I.
i
:c U
10
0
1
When wheat plants were treated with norflurazon during germination, the synthesis of carotenoids was inhibited. The carotenoid content of dark-grown plants was about
345
8
Number of flashes
Fig. 1: Accumulation of Chlide in flash (1-6 flashes) irradiated dark-grown plants. The plants were treated with 10 and 100 J.1mollL SAN-9789 during germination.
30 •
non·tr••tad plante
~ 10 Ilmoi/L SAN·9789
0100 Ilmoi/L SAN-1I789
o Darkn...
Results
2
1
2
3
4
5
8
Number of flashes Fig. 2: Photoreduction of Pchlide in flash (1-6 flashes) irradiated dark-grown plants. The Pchlide accumulated in darkness and the amount of Pchlide left over after flashes are present. The plants were treated with 10 and 100~ollL SAN-9789 during germination.
Chlorophyllide Accumulation in SAN-treated Plants
The rate of the phototransformation of Pchlide, initially accumulated in darkness, to Chlide in irradiated dark-grown plants is given as a ratio of Chlide/Pchlide in Table 1. After irradiation with an increasing number of flashes (1 to 6), the ratio Chlide/Pchlide increased from 0.63 to 1.79 in nontreated plants, from 0.83 to 2.28 at 10 J.lmollL SAN and from 1.16 to 3.00 at-100 J.lmollL SAN. In SAN-treated plants irradiated with 1 flash, the amount of Pchlide transformed to Chlide was larger than in non-treated plants, and this effect was seen also after irradiation with an increasing number of flashes. In order to explain the observed faster transformation of the originally accumulated Pchlide to Chlide in SAN-treated plants, the ratio between the photoactive and non-photoactive Pchlide was determined on the basis of the low-temperature fluorescence emission spectra of detached leaves (Fig. 3 a, b). In the dark-grown plants the ratio Pchlide657/Pchlide633 was 3.69 in the non-treated and 3.78 in the SAN-treated ones (10 J.lmoI/L) after their irradiation with 5 flashes the ratio Chlide694/Pchlide657+ Pchlide633 was 2.3 and 2.9, respectively (Table2). It was found before (Klockare et al., 1981; Minkov and Jahoubian, 1993) that the total amount of originally accumulated Pchlide was higher in dark-grown carotenoid-deficient plants. To observe the effect of SAN-treatment on the resynthesis of Pchlide, dark-grown plants irradiated with 5 flashes
1 2 3 4 5 6
Non-treated plants
0.63** 0.80** 1.11** 1.57** 1.69* 1.79
a non-treated plants ....... SAN-treated plants
II)
c u
II)
en
3
.... I)
0
:::s 2
... I)
>
'" I)
a:
4 II)
u c II) 3 u en
non-treated plants ...... SAN-treated plants
SAN-treated plants 10 ~mollL
100 ~mollL
0.83** 0.99** 1.24** 1.69** 2.00* 2.28
1.16** 1.69** 1.84** 2.04** 3.17* 3.00
Table 2: Influence of SAN-9789 (10 ~ollL) on the photoreduction of Pchlide to Chlide, presented as ratios of different pigment forms before and after 5 flashes irradiation of etiolated leaves. The ratios were determined on the basis of the pigment maxima of the lowtemperature fluorescence spectra of detached leaves. The excitation wavelength was set at 440 nm. Non-treated plants
SAN-treated plants
PchlidC657/Pchlide633 in dark-grown plants
3.69
3.78
Chlide657/Pchlide657 + Pchlide633 in flash irradiated plants
2.20
2.90
..
b
.... 0
II)
:::s ;: 2
II)
.~
cv
a;
a:
0 600
l
Table 1: Influel'le of SAN-9789 on the photoreduction of Pchlide to Chlide (presented as a ratio Chlide/Pchlide) in flash-irradiated dark-grown plants. The ratios were determined on the basis of the pigment maxima of the fluorescence emission spectra in acetone extracts. The excitation wavelength was set at 440 nm. The mean values and the mean derivations are given according to Student; *p<0.05; **p
4
651
650
700
750
Wavelength. nm Fig.3: Low-temperature fluorescence emission spectra of etiolated leaves (a); after irradiation with 5 flashes (b). The excitation wavelength - 440 nm. The spectra were normalised at their maxima at 633 nm. The plants were treated with 10 and 100 ~mollL SAN-9789 during germination.
were left in darkness for 60 min (Fig. 4). After the 30th min of redarkening, there was no difference in Pchlide reaccumulation between non-treated and treated plants. From the 30th min to the 60th min the amount of Pchlide strongly increased in SAN-treated plants compared with non-treated ones. Discussion
The treatment of plants with SAN-9789 causes an inhibition of the carotenoid synthesis (Bartels and McCullough, 1972; Klockare et al., 1981). When SAN-treated plants are grown in darkness the synthesis of Pchlide is stimulated. During the first hour of greening of carotenoid-deficient plants the accumulation of Chl is also stimulated but after that the Chl content gradually decreases (Minkov and Jahoubjan, 1993). The decrease of ChI could be a secondary process caused by photooxidation as a result of the disturbed photoprotective function of carotenoids, but the enhanced accumulation of Chl and its precursors needs further explanation. In our experiIl1ents we irradiated dark-grown carotenoidcontaining and carotenoid-deficient wheat plants with a dif-
652
-
GAUNA T. }AHOUBJAN and IVAN N. MINKOW
treatment. Consequently, the higher total amount of the originally accumulated Pchlide reflects the enhanced accumula---.. non-treated plants tion of the non-photoactive as well as of the photoactive 'j • - . 10 Ilmoi/L SAN-9789 forms of Pchlide in the carotenoid-deficient plants. Neverthe.c less, after the 5 flashes of irradiation, the ratio Chlide6941 lit Pchlid<=657+ Pchlide633 was higher in SAN-treated plants compared with non-treated plants (Table 2; Fig. 3 a, b), and it 10 j' confirms the assumption that a greater number of Pchlide Q molecules are phototransformed to Chlide in flash-irradiated Q :::I. SAN-treated plants. We have found that the accumulation of Chl was faster after the irradiation of dark-grown carotenoid:S! deficient plants with weak red light (Minkov and Jahoubjan, ::&: u 1993). In such conditions the light screening does not dea.. pend so strongly on the decreased carotenoid content, so the 5 30 o 60 observed effects of SAN-treatment might be also due to some changes of inner etioplast membrane conformation, caused by the missing carotenoids. There are, however, some data Time of re-darkness, min that the lack of carotenoids does not change the ultrastrucFig.4: Reaccumulation of Pchlide in redarkened plants after irradia- ture of the PLBs in SAN-treated leaves (Ryberg et al., 1981). tion with 5 flashes. The plants were treated with 10 I1mollL SANThe enhanced accumulation of Chlide, resp. Chl, during 9789 during germination. the first hour of greening of dark-grown SAN-treated plants (Minkov and Jahoubjan, 1993) could also be caused by a stimulated resynthesis of Pchlide. Klockare et al. (1981) ferent number of flashes (1 to 6). The total light exposure af- showed a greater total amount of Pchlide in dark-grown ter 6 flashes was enough for photo transformation of Pchlide SAN-treated plants, but its resynthesis was inhibited as a reto Chlide to take place, but not for the esterification of sult of PLBs damage when the plants were grown at continuChlide to Chl, which occurs between 10 and 60 min (laf- ous weak red light. Our results showed that the SAN-treatleche et al., 1972), so here we mainly discuss Chlide, but not ment stimulated the resynthesis of Pchlide in redarkened Chl accumulation. At the same time this way of irradiation plants that had been flash-irradiated, but it took 30 min lagprotected the carotenoid-deficient plants from photodynamic phase (Fig. 4). We can conclude that the enhanced ChI accumulation in damage, which usually takes place after longer irradiation of such plants. This was confirmed by the higher Chlide con- SAN-treated dark-grown plants can be achieved by flash irratent in irradiated, SAN-treated plants (Fig: 1). Nevertheless, diation or a brief irradiation with weak light. Under these the possibility of a limited effect of a small amount of singlet conditions the observed effect is a result of a stimulated synthesis and resynthesis of Chl precursors, as well as of a photooxygen could not be totally excluded. The Chlide accumulation and the Pchlide reduction were transformation of a greater number of Pchlide molecules per faster in SAN-treated plants, irradiated with an increasing time unit in the SAN-treated plants. Consequently, the Chl number of flashes compared with the non-treated ones. It is synthesis that had been stopped by darkness can be continclear from Fig. 1 and Fig. 2 that the enhanced formation of ued more efficiently in flash-irradiated carotenoid-deficient Chlide in norflurawn-treated plants reflects the higher plants compared with non-treated ones. Pchlide content that had already been accumulated in darkness. On the other hand, the lack of carotenoids probably Acknowledgements causes phototransformation of a greater number of Pchlide This investigation was supported by the Foundation of Fundamolecules per time unit in SAN-treated plants at unsaturated light conditions. This is confirmed by the phototransforma- mental Research of Belarus and the Bulgarian National Science tion of Pchlide to Chlide (Table 1), where concomitantly Fond, Grants K-411 and MU-HP-3. with the increasing flash number (1-6) and establishment of saturated light conditions, the ratio Chlide\Pchlide increased 1.16, 1.45 and 1.84 times in non-treated, 10 and 100llmollL References SAN-treated plants, respectively. In this way the light screening effect of the carotenoids is negligible in carotenoid-defi- ADAMSKA, I., K. KwPPSTECH, and I. OHAD: UV light stress induces the synthesis of the early light-inducible protein and prevents its cient plants. Our findings confirm the statements of Koski et degradation.}. BioI. Chem. 267, 34, 24732-24737 (1992). al. (1951) that the carotenoids depress the transformation of protochlorophyll to chlorophyll in normal etiolated corn AxELSSON, L., B. KwcKARE, H. RYBERG, and A.-S. SANDEUUS: In: AKOYUNOGLU, G. (ed.): Photosynthesis Vol. V. Chloroplast deleaves because they absorb blue light very strongly, and that velopment, pp. 285-293. Balaban international Science Services, the lower efficiency of the photochemical action in normal Philadelphia, PA (1981). seedlings, compared with albino seedlings, can be attributed to AxELSSON, L., c. DAHUN, and H. RYBERG: The function of carotcompetitive absorption of blue light by carotenoid pigments. enoids during chloroplast development. V. Correlation between In the dark-grown plants the ratio Pchlid<=657/Pchlide633 carotenoid content, ultrastructure and chlorophyll b to chlorowas nearly equal (Table 2), irrespective of the norflurawnphyll a ratio. Physiol. Plant. 55, 111-116 (1982). .Qc
•.,
-. .,-
15
Chlorophyllide Accumulation in SAN-treated Plants BARTELS, P. G. and C. MCCULLOUGH: A new inhibitor of carotenoid synthesis in higher plants: 4-chloro-5-(dimethylamino)2-N,N,N(trifluoro-m-tolyl)-3(2H)-pyridazinon (Sandoz 6706). Biochem. Biophys. Res. Comm. 48, 16-22 (1972). DAHLIN, c., H. RYBERG, and L. AxELSSON: A possible structural role for carotenoids and carotenoid precursors in etioplasts. Physiol. Plant. 59. 562-566 (1983). DUJARDIN, E.: Emerson-like effect in protochlorophyll(ide) photoreduction in bean leaves. In: METZNER, H. (00.): Photosynthetic Oxygen Evolution, pp. 511-520. Academic Press, New York (1978). DUJARDIN, E., A. KAHN, and P. MATHIS: Soret absorption of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction trapped at low temperature. Physiol. Plant. 78, 123-127 (1990). lKEUCHI, M. and S. MURAKAMI: Separation and characterization of prolamellar bodies and prothylakoids from squash etioplasts. Plant Cell Physiol. 24,71-80 (1983). KAHN, A, N. K. BOARDMAN, and S. W. THORNE: Energy transfer between protochlorophyllide molecules: evidence for multiple chromophores in the photoactive protochlorophyllide-protein complex in vivo and in vitro. J. Mol. BioI. 48.85 (1970). KAHN, A.: Spectrophotometric quantitation of protochlorophyll (ide): Specific absorption and molar extinction coefficients reconsidered. Physiol. Plant. 59. 99-102 (1983). KLocKARE, B., L. AxELSSON, H. RYBERG, A. S. SANDELIUS, and K.O. WIDELL: The function of carotenoids during chloroplast development. I. Effects of the herbicide SAN-9789 on chlorophyll synthesis and plastid ultrastructure. In: AKOYUNOGwu, G. (ed.): Photosynthesis. Vol. V, pp. 277-284. Balaban Int. Sci. Servo Philadelphia (1981). KOSKI, V. M., C:S. FRENCH, and J. H. C. SMITH: The Action Spectrum for the Transformation of Protochlorophyll to Chlorophyll a in Normal and Albino Corn Seedlings. Arh. Biochem. Biophys., Vol. 31. N 1, 1-15 (1951). KRINSKY, N. I.: The protective function of carotenoid pigments. In: GIESE, A. C. (ed.): Photophysiol. Curro Top., Vol. IlL pp. 123195. Acad. Press (1968).
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LAFLECHE, D., J. M. BOVE, and J. DURANToN: Localization and translocation of the protochlorophyllide holochrome during the greening of etioplasts in Zea mays L. J.ltrastruct. Res. 40.205 (1972), LINDSTEN, A, M. RYBERG, and C. SUNDQVIST: The polypeptide composition of highly purified pro lamellar bodies and prothylakoids from wheat (Triticum IUstivum) as revealed by silver staining. Physiol. Plant. 72. 167-176 (1988). LITVIN, E E, E. I. EFIMTSEV, N. V. IGNATov, and o. B. BELYAEVA: Confirmation of the existence of two photochemical reactions during biosynthesis of Chl and investigation of energy transfer between them. Fiziol. Rast. (Moscow) 23, 17-24 (in Russian) (1976). LITVIN, E E, N. V. IGNATov, and o. B. BELYAEVA: Photoreversibility of transformation of protochlorophyllide into chlorophyllide. Photobiochem. Photobiophys. 2, 233 (1981). MACKrNNEY, G. J.: Absorption of light by chlorophyll solution. BioI. Chern. 140,315-322 (1941). MINKOV, I. and G. JAHOUBJAN: Influence of SAN-9789 on the chlorophyll accumulation and its precursors in wheat plants. Bulg. J. Plant Phys. 19,45-51 (1993). RYBERG, H., L. AxELSSON, B. KLoCKARE, and A S. SANDELIUS: The function of carotenoids during chloroplast development. III. Protection of the prolamellar body and the enzymes chlorophyll synthesis from photodestruction sensitized and by early forms of chlorophyll, in Photosynthesis, V. Chloroplast Development, AKOYUNOGWU, G. Ed., pp. 295-304. Balaban Int. Science Services, Philladelphia, PA (1981). RYBERG, M. and C. SUNDQVIST: Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts. Physiol. Plant. 56, 125 (1982a). - - Spectral forms of protochlorophyllide in prolamellar bodies and prothylakoids fractionated from wheat etioplasts. Physiol. Plant. 56, 133 (1982b). VIRGIN, H. I., A KAHN, and D. WETTSTEIN: The physiology of Chl formation in relation to structural changes in chloroplasts. Photochem. Photobiol. 2, 83-91 (1963). VIRGIN, H. I.: The physical state of protochlorophyll(ide) in plants. Ann. Rev. Plant. Physiol. 32,451-463 (1981).
• ••••• AL.F. © 1997 by Gustav Fischer Verlag, Jena
Enhanced Chlorophyllide Accumulation after Flash Irradiation of Etiolated Wheat Plants Treated with SAN-9789 GAUNA T. ]AHOUBJAN
and
IVAN
N.
MINKOV
University of Plovdiv, Plant Physiology Department, Tsar Assen 24 St., 4000 Plovdiv, Bulgaria Received February 24, 1997 . Accepted June 18, 1997
Summary
The accumulation of Chlide was investigated in flash-irradiated dark-grown plants as dependent on the decreased carotenoid content. Wheat plants (Triticum aestivum L.) were treated with two concentrations (10 and 100 ~mol/L) of norflurazon (SAN-9789) during germination, which caused inhibition of carotenoid synthesis. When SAN-treated plants were grown in darkness the amount of aCCJ,lmulated protochlorophyllide increased. After an irradiation of carotenoid-deficient etiolated plants with an increasing number of flashes (1-6) the decrease of protochlorophyllide and the accumulation of chlorophyllide were faster in comparison with the non-treated ones. The flash irradiation of carotenoid-deficient plants caused phototransformation of a greater number of originally accumulated protochlorophyllide molecules to chlorophyllide, and in that way the ChI synthesis that had been stopped in darkness could continue more efficiently.
Kry words: Wheat, Triticum aestivum L., protochlorophyllide, chlorophyllide, SAN-9789 (norjlurazon), flash irradiation. Abbreviations: Chl = chlorophyll; Chlide = chlorophyllide; Pchlide = protochlorophyllide; SAN-9789 = norflurazon; PLBs =prolamellar bodies; PTs = prothylakoids. Introduction
In dark-grown angiosperms the proplastids develop into etioplasts instead of chloroplasts, and protochlorophyllide (Pchlide) accumulates as a main plastid pigment (Virgin et al., 1963, 1981). Pchlide-protein complexes characterised by their fluorescence and absorption spectra are present in at least three spectral forms in inner etioplast membranes: Pchlid~50_657 and Pchlid~36-657' localised mainly in the prolamellar bodies (PLBs) (Ryberg and Sundqvist, 1982 a, b; Ikeuchi and Murakami, 1983; Lindsten et al., 1988), and Pchlid~28_633' localised mainly in the prothylakoids (PTs) (Lindsten et al., 1988). The irradiation of etiolated plants induces a transformation of Pchlide650-657 and Pchlid~36-657 to chlorophyllide (Chlide), whereas Pchlid~28-633 is considered a non-phototransformable form (Kahn et al., 1970). The photoconversion of Pchlide to Chlide involves a formation of j. Plant PhysioL Wll. 151. pp. 649-653 (1997)
several Chlide forms (Litvin et al., 1976, 1981; Dujardin, 1978, 1990). The treatment of plants with norflurazon (SAN-9789) causes an inhibition of the carotenoid synthesis, and the precursors phytoene and phytofluene accumulate (Bartels and McCullough, 1972). The effect of SAN-9789 on the plastids could be connected to the photoprotective role of carotenoids for chlorophyllide sensitised photodestruction (Krinsky, 1968; Ryberg et al., 1981) or to their structural role (Axelsson et al., 1981, 1982; Dahlin et al., 1983). When plants are grown in darkness, the total content of Pchlide is increased in SAN-treated plants. There is no structural difference in the PLBs between non-treated and treated plants (Klockare et al., 1981). This tendency is kept immediately after the phototransformation of Pchlide (1 s, week red light). During the following dark period (1 to 3 h) the photodecomposition of Chlide and the structural changes in PLBs
650
GAUNA T. JAHOUBJAN and IVAN N. MINKOW
occur faster in the treated leaves than in carotenoid-containing ones (Klockare et al., 1981; Ryberg et al., 1981). When carotenoid-deficient plants develop in light, the early lightinducible proteins (ELIPs) are specifically induced (Adamska et al., 1992). According to Axellson et al. (1981, 1982), the Chi content in plants grown under continuous red light at higher SAN9789 concentration is lower and is due to the inhibition of Chi synthesis and the faster decomposition of the Chi formed. On the other hand, we have previously shown (Minkov and Jahoubjan, 1993) that the total Chi content was higher during the first hour of irradiation (weak red or white light) of dark-grown SAN-treated plants compared with nontreated ones, which was followed by a decrease of Chi, most probably due to a progressing photodecomposition. The reason for the increased Chi content in greening carotenoiddeficient leaves is not known. In this paper we investigated the accumulation of ChIide in flash-irradiated dark-grown plants treated with SAN-9789 in dependence of the decreased carotenoid content, as well as the increased accumulation and re-accumulation ofPchIide in darkness. Material and Methods
Plant mauritz! Seeds of wheat (Triticum aestivum L., cv. Pobeda) were soaked for 24 h in water, or in water containing SAN-9789 (Sandoz LTD, Switzerland) in two different concentrations, i.e. 10 and 100 ~oll L. The seedlings were grown in darkness at 25 ·C for 8 days. The Pchlide in the dark-grown leaves was phototransformed to Chlide by a different number of flashes (1 ens of «white light» followed by 5 s of darkness, impulse energy 120 J) at a distance of 20 em.
Pigment analysis The contents of Pchlide, Chlide and carotenoids were determined in 85 % acetone. The extracts were measured fluorometricaIly on an LS-3B Perkin-Elmer spectrofluorometer and their emission spectra (600-750 nm) were recorded at an excitation wavelength of 440 nm. The pigment content was calculated using the molar extinction coefficients of Kahn (1983) for Pchlide and of MacKinney (1941) for Chlide. The phototransformation of Pchlide to Chlide is presented as a ratio Chlide/Pchlide, calculated on the basis of their fluorescence maxima in acetone extracts. The carotenoid content was determined according to MacKinney (1941). The mean values were based on 12 repetitions with 10 plants in each one. The mean deviations (SE) were calculated according to Student and only the values with p<0.05 were discussed. The low-temperature emission spectra (600-750 nm; excitation wavelength 440 nm) of the detached leaves were recorded at 77 K before and after flash irradiation. The spectra were normalised at the same value at 633 nm. On the basis of these spectra the ratios PchlidC(;57/PchlidC(;33 and ChlidC(;95/PchlidC(;57+ PchlidC(;33 were calculated.
220 ~ g-1 &esh weight in non-treated leaves, whereas it was 93 and 34/J.gg-1 at treatment with 10 and 100/J.mo1lL SAN9789, respectively. The SAN-treatment had no effect on the water content and the dry weight of etiolated and briefly irradiated plants. The latter varied about 11 % in non-treated and treated plants. After irradiation of the dark-grown plants with a different number of flashes, higher amounts of ChIide accumulated in the carotenoid-deficient plants compared with the non-treated plants. In the course of flash irradiation (1 to 6) the ChIide content increased from 9.6 to 18.1 /J.gg-1 fresh weight in non-treated plants, from 12.9 to 25.8 /J.g g-l at 10/J.mo1lL SAN and &om 17.1 to 29.7/J.gg-1 at 100/J.mo1lL SAN (Fig. 1). After irradiation of the dark-grown plants with 6 flashes, the Pchlide content decreased 3.4, 4.0 and 5.2 times in non-treated, 10 and 100/J.M SAN-treated plants, respectively {Fig. 2).
-
•
non·treated plante
~ 10 Ilmoi/L SAN·9789
a 30
o 100 Ilmoi/L SAN·9789
'i it .c
., • 20
.t:
0;-
at at
::I.
i
:c U
10
0
1
When wheat plants were treated with norflurazon during germination, the synthesis of carotenoids was inhibited. The carotenoid content of dark-grown plants was about
345
8
Number of flashes
Fig. 1: Accumulation of Chlide in flash (1-6 flashes) irradiated dark-grown plants. The plants were treated with 10 and 100 J.1mollL SAN-9789 during germination.
30 •
non·tr••tad plante
~ 10 Ilmoi/L SAN·9789
0100 Ilmoi/L SAN-1I789
o Darkn...
Results
2
1
2
3
4
5
8
Number of flashes Fig. 2: Photoreduction of Pchlide in flash (1-6 flashes) irradiated dark-grown plants. The Pchlide accumulated in darkness and the amount of Pchlide left over after flashes are present. The plants were treated with 10 and 100~ollL SAN-9789 during germination.
Chlorophyllide Accumulation in SAN-treated Plants
The rate of the phototransformation of Pchlide, initially accumulated in darkness, to Chlide in irradiated dark-grown plants is given as a ratio of Chlide/Pchlide in Table 1. After irradiation with an increasing number of flashes (1 to 6), the ratio Chlide/Pchlide increased from 0.63 to 1.79 in nontreated plants, from 0.83 to 2.28 at 10 J.lmollL SAN and from 1.16 to 3.00 at-100 J.lmollL SAN. In SAN-treated plants irradiated with 1 flash, the amount of Pchlide transformed to Chlide was larger than in non-treated plants, and this effect was seen also after irradiation with an increasing number of flashes. In order to explain the observed faster transformation of the originally accumulated Pchlide to Chlide in SAN-treated plants, the ratio between the photoactive and non-photoactive Pchlide was determined on the basis of the low-temperature fluorescence emission spectra of detached leaves (Fig. 3 a, b). In the dark-grown plants the ratio Pchlide657/Pchlide633 was 3.69 in the non-treated and 3.78 in the SAN-treated ones (10 J.lmoI/L) after their irradiation with 5 flashes the ratio Chlide694/Pchlide657+ Pchlide633 was 2.3 and 2.9, respectively (Table2). It was found before (Klockare et al., 1981; Minkov and Jahoubian, 1993) that the total amount of originally accumulated Pchlide was higher in dark-grown carotenoid-deficient plants. To observe the effect of SAN-treatment on the resynthesis of Pchlide, dark-grown plants irradiated with 5 flashes
1 2 3 4 5 6
Non-treated plants
0.63** 0.80** 1.11** 1.57** 1.69* 1.79
a non-treated plants ....... SAN-treated plants
II)
c u
II)
en
3
.... I)
0
:::s 2
... I)
>
'" I)
a:
4 II)
u c II) 3 u en
non-treated plants ...... SAN-treated plants
SAN-treated plants 10 ~mollL
100 ~mollL
0.83** 0.99** 1.24** 1.69** 2.00* 2.28
1.16** 1.69** 1.84** 2.04** 3.17* 3.00
Table 2: Influence of SAN-9789 (10 ~ollL) on the photoreduction of Pchlide to Chlide, presented as ratios of different pigment forms before and after 5 flashes irradiation of etiolated leaves. The ratios were determined on the basis of the pigment maxima of the lowtemperature fluorescence spectra of detached leaves. The excitation wavelength was set at 440 nm. Non-treated plants
SAN-treated plants
PchlidC657/Pchlide633 in dark-grown plants
3.69
3.78
Chlide657/Pchlide657 + Pchlide633 in flash irradiated plants
2.20
2.90
..
b
.... 0
II)
:::s ;: 2
II)
.~
cv
a;
a:
0 600
l
Table 1: Influel'le of SAN-9789 on the photoreduction of Pchlide to Chlide (presented as a ratio Chlide/Pchlide) in flash-irradiated dark-grown plants. The ratios were determined on the basis of the pigment maxima of the fluorescence emission spectra in acetone extracts. The excitation wavelength was set at 440 nm. The mean values and the mean derivations are given according to Student; *p<0.05; **p
4
651
650
700
750
Wavelength. nm Fig.3: Low-temperature fluorescence emission spectra of etiolated leaves (a); after irradiation with 5 flashes (b). The excitation wavelength - 440 nm. The spectra were normalised at their maxima at 633 nm. The plants were treated with 10 and 100 ~mollL SAN-9789 during germination.
were left in darkness for 60 min (Fig. 4). After the 30th min of redarkening, there was no difference in Pchlide reaccumulation between non-treated and treated plants. From the 30th min to the 60th min the amount of Pchlide strongly increased in SAN-treated plants compared with non-treated ones. Discussion
The treatment of plants with SAN-9789 causes an inhibition of the carotenoid synthesis (Bartels and McCullough, 1972; Klockare et al., 1981). When SAN-treated plants are grown in darkness the synthesis of Pchlide is stimulated. During the first hour of greening of carotenoid-deficient plants the accumulation of Chl is also stimulated but after that the Chl content gradually decreases (Minkov and Jahoubjan, 1993). The decrease of ChI could be a secondary process caused by photooxidation as a result of the disturbed photoprotective function of carotenoids, but the enhanced accumulation of Chl and its precursors needs further explanation. In our experiIl1ents we irradiated dark-grown carotenoidcontaining and carotenoid-deficient wheat plants with a dif-
652
-
GAUNA T. }AHOUBJAN and IVAN N. MINKOW
treatment. Consequently, the higher total amount of the originally accumulated Pchlide reflects the enhanced accumula---.. non-treated plants tion of the non-photoactive as well as of the photoactive 'j • - . 10 Ilmoi/L SAN-9789 forms of Pchlide in the carotenoid-deficient plants. Neverthe.c less, after the 5 flashes of irradiation, the ratio Chlide6941 lit Pchlid<=657+ Pchlide633 was higher in SAN-treated plants compared with non-treated plants (Table 2; Fig. 3 a, b), and it 10 j' confirms the assumption that a greater number of Pchlide Q molecules are phototransformed to Chlide in flash-irradiated Q :::I. SAN-treated plants. We have found that the accumulation of Chl was faster after the irradiation of dark-grown carotenoid:S! deficient plants with weak red light (Minkov and Jahoubjan, ::&: u 1993). In such conditions the light screening does not dea.. pend so strongly on the decreased carotenoid content, so the 5 30 o 60 observed effects of SAN-treatment might be also due to some changes of inner etioplast membrane conformation, caused by the missing carotenoids. There are, however, some data Time of re-darkness, min that the lack of carotenoids does not change the ultrastrucFig.4: Reaccumulation of Pchlide in redarkened plants after irradia- ture of the PLBs in SAN-treated leaves (Ryberg et al., 1981). tion with 5 flashes. The plants were treated with 10 I1mollL SANThe enhanced accumulation of Chlide, resp. Chl, during 9789 during germination. the first hour of greening of dark-grown SAN-treated plants (Minkov and Jahoubjan, 1993) could also be caused by a stimulated resynthesis of Pchlide. Klockare et al. (1981) ferent number of flashes (1 to 6). The total light exposure af- showed a greater total amount of Pchlide in dark-grown ter 6 flashes was enough for photo transformation of Pchlide SAN-treated plants, but its resynthesis was inhibited as a reto Chlide to take place, but not for the esterification of sult of PLBs damage when the plants were grown at continuChlide to Chl, which occurs between 10 and 60 min (laf- ous weak red light. Our results showed that the SAN-treatleche et al., 1972), so here we mainly discuss Chlide, but not ment stimulated the resynthesis of Pchlide in redarkened Chl accumulation. At the same time this way of irradiation plants that had been flash-irradiated, but it took 30 min lagprotected the carotenoid-deficient plants from photodynamic phase (Fig. 4). We can conclude that the enhanced ChI accumulation in damage, which usually takes place after longer irradiation of such plants. This was confirmed by the higher Chlide con- SAN-treated dark-grown plants can be achieved by flash irratent in irradiated, SAN-treated plants (Fig: 1). Nevertheless, diation or a brief irradiation with weak light. Under these the possibility of a limited effect of a small amount of singlet conditions the observed effect is a result of a stimulated synthesis and resynthesis of Chl precursors, as well as of a photooxygen could not be totally excluded. The Chlide accumulation and the Pchlide reduction were transformation of a greater number of Pchlide molecules per faster in SAN-treated plants, irradiated with an increasing time unit in the SAN-treated plants. Consequently, the Chl number of flashes compared with the non-treated ones. It is synthesis that had been stopped by darkness can be continclear from Fig. 1 and Fig. 2 that the enhanced formation of ued more efficiently in flash-irradiated carotenoid-deficient Chlide in norflurawn-treated plants reflects the higher plants compared with non-treated ones. Pchlide content that had already been accumulated in darkness. On the other hand, the lack of carotenoids probably Acknowledgements causes phototransformation of a greater number of Pchlide This investigation was supported by the Foundation of Fundamolecules per time unit in SAN-treated plants at unsaturated light conditions. This is confirmed by the phototransforma- mental Research of Belarus and the Bulgarian National Science tion of Pchlide to Chlide (Table 1), where concomitantly Fond, Grants K-411 and MU-HP-3. with the increasing flash number (1-6) and establishment of saturated light conditions, the ratio Chlide\Pchlide increased 1.16, 1.45 and 1.84 times in non-treated, 10 and 100llmollL References SAN-treated plants, respectively. In this way the light screening effect of the carotenoids is negligible in carotenoid-defi- ADAMSKA, I., K. KwPPSTECH, and I. OHAD: UV light stress induces the synthesis of the early light-inducible protein and prevents its cient plants. Our findings confirm the statements of Koski et degradation.}. BioI. Chem. 267, 34, 24732-24737 (1992). al. (1951) that the carotenoids depress the transformation of protochlorophyll to chlorophyll in normal etiolated corn AxELSSON, L., B. KwcKARE, H. RYBERG, and A.-S. SANDEUUS: In: AKOYUNOGLU, G. (ed.): Photosynthesis Vol. V. Chloroplast deleaves because they absorb blue light very strongly, and that velopment, pp. 285-293. Balaban international Science Services, the lower efficiency of the photochemical action in normal Philadelphia, PA (1981). seedlings, compared with albino seedlings, can be attributed to AxELSSON, L., c. DAHUN, and H. RYBERG: The function of carotcompetitive absorption of blue light by carotenoid pigments. enoids during chloroplast development. V. Correlation between In the dark-grown plants the ratio Pchlid<=657/Pchlide633 carotenoid content, ultrastructure and chlorophyll b to chlorowas nearly equal (Table 2), irrespective of the norflurawnphyll a ratio. Physiol. Plant. 55, 111-116 (1982). .Qc
•.,
-. .,-
15
Chlorophyllide Accumulation in SAN-treated Plants BARTELS, P. G. and C. MCCULLOUGH: A new inhibitor of carotenoid synthesis in higher plants: 4-chloro-5-(dimethylamino)2-N,N,N(trifluoro-m-tolyl)-3(2H)-pyridazinon (Sandoz 6706). Biochem. Biophys. Res. Comm. 48, 16-22 (1972). DAHLIN, c., H. RYBERG, and L. AxELSSON: A possible structural role for carotenoids and carotenoid precursors in etioplasts. Physiol. Plant. 59. 562-566 (1983). DUJARDIN, E.: Emerson-like effect in protochlorophyll(ide) photoreduction in bean leaves. In: METZNER, H. (00.): Photosynthetic Oxygen Evolution, pp. 511-520. Academic Press, New York (1978). DUJARDIN, E., A. KAHN, and P. MATHIS: Soret absorption of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction trapped at low temperature. Physiol. Plant. 78, 123-127 (1990). lKEUCHI, M. and S. MURAKAMI: Separation and characterization of prolamellar bodies and prothylakoids from squash etioplasts. Plant Cell Physiol. 24,71-80 (1983). KAHN, A, N. K. BOARDMAN, and S. W. THORNE: Energy transfer between protochlorophyllide molecules: evidence for multiple chromophores in the photoactive protochlorophyllide-protein complex in vivo and in vitro. J. Mol. BioI. 48.85 (1970). KAHN, A.: Spectrophotometric quantitation of protochlorophyll (ide): Specific absorption and molar extinction coefficients reconsidered. Physiol. Plant. 59. 99-102 (1983). KLocKARE, B., L. AxELSSON, H. RYBERG, A. S. SANDELIUS, and K.O. WIDELL: The function of carotenoids during chloroplast development. I. Effects of the herbicide SAN-9789 on chlorophyll synthesis and plastid ultrastructure. In: AKOYUNOGwu, G. (ed.): Photosynthesis. Vol. V, pp. 277-284. Balaban Int. Sci. Servo Philadelphia (1981). KOSKI, V. M., C:S. FRENCH, and J. H. C. SMITH: The Action Spectrum for the Transformation of Protochlorophyll to Chlorophyll a in Normal and Albino Corn Seedlings. Arh. Biochem. Biophys., Vol. 31. N 1, 1-15 (1951). KRINSKY, N. I.: The protective function of carotenoid pigments. In: GIESE, A. C. (ed.): Photophysiol. Curro Top., Vol. IlL pp. 123195. Acad. Press (1968).
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LAFLECHE, D., J. M. BOVE, and J. DURANToN: Localization and translocation of the protochlorophyllide holochrome during the greening of etioplasts in Zea mays L. J.ltrastruct. Res. 40.205 (1972), LINDSTEN, A, M. RYBERG, and C. SUNDQVIST: The polypeptide composition of highly purified pro lamellar bodies and prothylakoids from wheat (Triticum IUstivum) as revealed by silver staining. Physiol. Plant. 72. 167-176 (1988). LITVIN, E E, E. I. EFIMTSEV, N. V. IGNATov, and o. B. BELYAEVA: Confirmation of the existence of two photochemical reactions during biosynthesis of Chl and investigation of energy transfer between them. Fiziol. Rast. (Moscow) 23, 17-24 (in Russian) (1976). LITVIN, E E, N. V. IGNATov, and o. B. BELYAEVA: Photoreversibility of transformation of protochlorophyllide into chlorophyllide. Photobiochem. Photobiophys. 2, 233 (1981). MACKrNNEY, G. J.: Absorption of light by chlorophyll solution. BioI. Chern. 140,315-322 (1941). MINKOV, I. and G. JAHOUBJAN: Influence of SAN-9789 on the chlorophyll accumulation and its precursors in wheat plants. Bulg. J. Plant Phys. 19,45-51 (1993). RYBERG, H., L. AxELSSON, B. KLoCKARE, and A S. SANDELIUS: The function of carotenoids during chloroplast development. III. Protection of the prolamellar body and the enzymes chlorophyll synthesis from photodestruction sensitized and by early forms of chlorophyll, in Photosynthesis, V. Chloroplast Development, AKOYUNOGWU, G. Ed., pp. 295-304. Balaban Int. Science Services, Philladelphia, PA (1981). RYBERG, M. and C. SUNDQVIST: Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts. Physiol. Plant. 56, 125 (1982a). - - Spectral forms of protochlorophyllide in prolamellar bodies and prothylakoids fractionated from wheat etioplasts. Physiol. Plant. 56, 133 (1982b). VIRGIN, H. I., A KAHN, and D. WETTSTEIN: The physiology of Chl formation in relation to structural changes in chloroplasts. Photochem. Photobiol. 2, 83-91 (1963). VIRGIN, H. I.: The physical state of protochlorophyll(ide) in plants. Ann. Rev. Plant. Physiol. 32,451-463 (1981).