The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m²) was investigated for the chlorophyll(ide) pigments formed after photoreduction of the protochlorophyllide in dark grown leaves of wheat. Connections were found between changes in absorption spectrum in vivo (the Shibata shift and the late red-shift) and changes in photostability both in young (five-day) and old (12-day) leaves. The photostability of both the 684-form and the 673-form as well as the rate of the changes in photostability (the Shibata shift and the late red-shift) decreased with the age of the dark grown plants. It was concluded that the more pronounced decrease in the chlorophyll(ide) contents found at irradiation of older dark grown leaves mostly depended on the lower rate of the changes in the photostability of the pigment in old leaves. No resynthesis of protochlorophyllide occurred before the onset of the late red-shift. The results and their connection with the lag in chlorophyll formation are discussed. This lag is more pronounced in older dark grown wheat.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m^?2) was investigated for the chlorophyll(ide) pigments formed after the primary photoreduction of the protochlorophyll(ide) in dark grown leaves of wheat. After photoreduction, most of the chlorophyll(ide) exists in a form with an absorption maximum at 684 nm. This form is gradually transformed into a form with an absorption maximum at 673 nm (the Shibata shift). It was possible to ascribe a specific photostability to each of the pigment forms. This photostability was higher for the 673-form than for the 684-form. A red-shift in the absorption maximum following upon the Shibata shift, reflects the successive transformation of the 673-form into other pigment forms, which were quite photostable at the intensity used.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The effect of denaturing treatments on the stability against high intensity irradiation (red light, 700 W m^?2) was investigated in vivo for various chlorophyll forms in wheat. Three pigment forms were investigated: the 650-form (protochlorophyllide) present in dark grown leaves; the 684-form (chlorophyllide) formed within 5 s after photoreduction of the 650-form; and the 673-form (chlorophyll), into which the 684-form has been transformed 25 min after photoreduction of the 650-form. (The pigment forms are denoted by their absorption maxima in the red region before denaturation.) Two denaturing treatments were used: heat treatment (water of 55°C for 2 min) and freezing and thawing (freezing in liquid nitrogen followed by thawing in water of 25°C). Heat treatment as well as freezing and thawing caused a shift in the absorption peak of the two nonesterified pigment forms. The peak of of the chlorophyllide 684-form shifted to 673 nm and that of the protochlorophyllide 650-form to 636 nm. The absorption maximum of the chlorophyll 673-form was not affected by the above treatments. Heat treatment as well as freezing and thawing had profound effects on the structural organization of the plastid pigments, as shown by a decrease in the photostability. For the 684-form, heat treatment reduced the photostability by a factor of about 14 (half-life in strong light changed from 170 s to 12 s). Freezing and thawing also reduced the photostability, although the effect was less pronounced (c. 3–4 times decrease in half-life). Upon transformation of the chlorophyllide 684-form into the chlorophyll 673-form (the Shibata-shift) the pigments became less sensitive to light, and were no longer “aggregated” by heat treatment. The “aggregating” effect of freezing and thawing was still present after the Shibata shift. The results thus verify a clear difference in structural organization of the 684-form and the 673-form, since the two pigment forms were differently affected by heat treatment. The 650-form behaved similarly to the 684-form, although it appeared to be slightly less aggregated by heat treatment. — The decrease in photostability, caused by heat treatment of the 684-form, changed the kinetics for the photodecomposition from a first towards a second order reaction.     

Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.     

The Absence of Protochlorophyll(ide) Accumulation in Algae with Inhibited Chlorophyll Synthesis

Golenkinia, Chlorella protothecoides, and mutant C-2A′ of Scenedesmus were grown in darkness and on media in which chlorophyll synthesis is reduced significantly. The pigments were analyzed by spectrophotometry or by paper chromatography and compared with similar extracts from light-grown algae and dark-grown beans. No protochlorophyll(ide) was present in the dark-grown algae indicating that chlorophyll synthesis is blocked by a mechanism other than feedback regulation of aminolevulinic acid synthesis by protochlorophyll(ide) which has been proposed for flowering plants.     

The After-Effect of Water Stress on Chlorophyll Formation during Greening and the Levels of Abscisic Acid and Proline in Dark Grown Wheat Seedlings

Cut seedlings of wheat plants (Triticum aestivum L. cv. Starke II Weibull) between 6 and 7 days old were water stressed in darkness by exposing them to air of 35% relative humidity 2.5 to 20 h. This treatment resulted in a water potential of -11 bars in the leaves after 20 h. The leaves were then rewatered and irradiated. The chlorophyll formation that took place in fully turgid leaves during the greening was markedly decreased in the case of the water-stress pretreatmet. and especially the lag phase was prolonged. The longer the stress pretreatment the more evident was the subsequent effect on chlorophyll formation. However, no linear relationship was found between the amount of stress and the chlorophyll content. Protochlorophyllide regeneration from endogenously formed δ-aminolevulinic acid was markedly decreased even after the shortest water-stress period. However, protochlorophyllide accumulation from exogenously supplied δ-aminolevulinic acid was only slightly decreased following the water-stress pretreatment. Further more, the ratio of protochlorophyllide₆₅₀ to protochlorophyllide₆₂₈ was slightly reduced by the same conditions. During the stress period both abscisic acid and proline were accumulated in the leaves. The content of abscisic acid increased up to six times the normal level during water stress lasting for 20 h. The increase of proline was about three-fold for similar treatment. After rewatering the leaves the levels of both abscisic acid and proline rapidly declined and reached. 10 h later, the levels found in unstressed seedlings. The increase in abscisic acid during water stress associated with impaired chlorophyll metabolism suggested that the after-effect of water stress might be linked to chlorophyll metabolism through abscisic acid or some of its metabolites. The changes in proline content open the possibility that this substance could function as a reserve substance for the formation of chlorophyll after the discon tinuation of the stress.     

The Pool Size of Protochlorophyllide during Different Stages of Greening of Dark Grown Wheat Leaves

The pool size of protochlorophyllide in wheat leaves irradiated for 5 minutes to 6 hours was studied. Protochlorophyllide then accumulated in the dark, but the pool size of regenerated protochlorophyllide was considerably smaller in leaves irradiated for six hours than in leaves irradiated for 5 minutes. The decrease in pool size of regenerated protochlorophyllide was found to take place at the time when the chlorophyll formation had accelerated and reached the linear phase. The protochlorophyllide accumulated is the form with absorption maximum at 650 nm, which is phototransformed to chlorophyllide with maximum absorption at 684 nm. This species goes through the Shibata shift when formed even after 6 hours of irradiation. If leaves, irradiated for 1 or 6 hours, were fed with δ-amino-levulinic acid the protochlorophyllide synthesis was only 1.2 times faster in the leaves irradiated for 6 hours than in those irradiated for 1 hour. In the case of leaves fed with δ-amino-levulinic acid the absorption maximum of protochlorophyllide is at 636 nm and the absorption maximum of the chlorophyllide formed is at 672 nm.     

Chlorophyll Formation in the Dark II. Chlorophyll in Wheat Leaves Transplanted to Pine Megagametophytes

Chlorophyll production in wheat (Triticum vulgare) leaves takes place in the dark when wheat embryos are transplanted to the megagametophytes of black pine (Pinus nigra). The chlorophyll content in the wheat leaves is about half that in black pine cotyledons of the same age.     

Comparison of Light-dependent Oxygen Uptake, Protochlorophyll(ide)-650 Photoconversion, and Chlorophyll Disappearance in Wheat Etioplasts

Red light exposures given to dark-grown wheat seedlings (Triticum aestivum L.) prior to etioplast isolation reduced the ability of these organelles to consume O₂. The same preharvest red light exposures also decreased protochlorophyll(ide) content of etioplasts. In addition, regeneration of both O₂ uptake rates as well as protochlorophyll(ide) levels followed a parallel time course. These similarities suggested that photoconversion of protochlorophyll(ide)-650 to chlorophyll(ide) may mediate some process with O₂ as the electron acceptor. This process appears to involve photooxidation of nonphotoconvertible protochlorophyll(ide) as well as of newly formed chlorophyll(ide). This hypothesis is further supported by the observations that: (a) the in vitro light induced O₂ uptake phenomenon was observed in solubilized protochlorophyll(ide) holochrome preparations; and (b) photoinduced O₂ uptake was reduced to zero rate by light exposure time equivalent to that required for chlorophyll(ide) and nonphotoconvertible protochlorophyll(ide) destruction.     

Chlorophyll Synthesis from Protochlorophyll(ide) in Chill-Stressed Maize (Zea mays L.)

Illumination of aetiolated maize at temperatures lower than20 °C results in negligible accumulation of chlorophyll.Illumination of leaf tissue, previously incubated in 10 molm^–3 ALA in darkness, shows only a slight conversion ofprotochlorophyll(ide) to chlorophyll a and b at temperaturesless than 20 °C. A refined procedure for measuring photosynthesisby photo-acoustic spectroscopy in leaves that differ in chlorophyllcontent is presented. Studies of photosynthesis in aetiolatedseedlings illuminated at different temperatures by photo-acousticspectroscopy suggests that impairment of the chlorophyll pathwayis paralleled by an aberrant development of the thylakoid membrane. Key words: Protochlorophyll(ide), temperature, photo-acoustic spectroscopy, membrane biogenesis     

A Reversible Conversion of Phototransformable Protochlorophyll(ide)(656) to Photoinactive Protochlorophyll(ide)(656) by Hydrogen Sulfide in Etiolated Bean Leaves

The relationship of phototransformable protochlorophyll-(ide) to photoinactive protochlorophyll(ide) has been studied in the primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Subjecting the leaves to an atmosphere of H₂S causes an immediate loss of phototransformable protochlorophyll(ide)₆₅₀ and a simultaneous increase in photoinactive protochlorophyll(ide)₆₃₃. When such leaves are returned to air or N₂, the absorbance at 650 nm increases, whereas the absorbance at 633 nm decreases and photoactivity is restored. The reversion of protochlorophyll-(ide)₆₃₃ to protochlorophyll(ide)₆₅₀ is one-half complete in 3 minutes at 22 C in 8-day-old leaves. Ninety-five per cent recovery of protochlorophyll(ide)₆₅₀ is obtained when exposure to H₂S is less than 3 minutes in duration; longer periods reduce the reversion capacity proportionately. The leaves are relatively undamaged by brief exposures to H₂S, as judged by electron microscopy and by their ability to synthesize chlorophyll under continuous illumination. Hydrogen sulfide has no immediate effect upon the absorption properties of a partially purified preparation of the protochlorophyll(ide) holochrome, an etioplast suspension, or leaves subjected to freezing and thawing. Compounds such as HCN and HN₃ cause an irreversible conversion of protochlorophyll(ide)₆₅₀ to protochlorophyll(ide)₆₃₃ with total loss of photoactivity. Sulfhydryl agents, such as β-mercaptoethanol and cysteine, cause a slow, irreversible transformation of the photoactive pigment to the photoinactive form and inhibit the ability of the leaves to synthesize chlorophyll under continuous illumination. The results obtained suggest that H₂S may alter the interaction between the source of hydrogens on the protein moiety of the holochrome and the chromophore in vivo by reducing a disulfide bond in the protein, thereby causing a reversible conformational change in the complex.     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells I. Chlorophyll Accumulation and Phototransformation of Protochlorophyll(ide) in Callus Cells under Blue and Red light

A marked accumulation of chlorophyll was observed in calluscells of Nicotiana glutinosa when they were grown under bluelight, while under strong red light no chlorophyll accumulated.This blue light effect saturated at an intensity of about 500mW.m^–2. The effects of white, blue and red light on the transformationof protochlorophyll (ide) (Pchl) accumulated in dark-grown calluscells were studied by following the changes in the intensityof fluorescence emitted by Pchl and different forms of chlorophyll(ide) (Chi). Pchl with a fluorescence maximum at 633 nm (absorptionmaximum: 630 nm) decreased slowly, concomitant with an increasein Chl having a fluorescence maximum at 677 nm (absorption maximum:675 nm), which was subsequently transformed, independently oflight, to Chi with a fluorescence maximum at 683 nm (absorptionmaximum: 680 nm). Both blue and red light of low intensitieswere effective for the phototransformation, while red light,but not blue light, of high intensities caused significant destructionof Pchl. An action spectrum for this photodestruction showedthat the maximum destruction took place at 630 nm. White lightof high intensities was effective for the photoreduction withonly slight destruction of Pchl, suggesting that blue lightcounteracts the destructive effect of red light. At low temperatures,however, blue light as well as red light of low intensitiescaused photodestruction of Pchl. It was inferred that blue lightenhances a certain step or steps involved in the productionof a reductant required for the photoreduction of Pchl to Chl. (Received July 3, 1981; Accepted November 11, 1981)     

Regulation of Protochlorophyll(ide) Levels in Dark-Grown Non-Dividing Euglena 3. Proplastid Structure during Loss and Regeneration of Protochlorophyll(ide)

Cells of Euglena gracilis Klebs var. bacillaris Cori growingin darkness on a complete medium have small undifferentiatedproplastids. On transfer to an incomplete (resting) medium indarkness, the cells cease division within 72 h. During thistime the proplastid expands and several prothylakoids and prolamellarbodies develop even though phototransformable protochlorophyll(ide)[PT-Pchl(ide)] is decreasing. As PT-Pchl(ide) decreases furtherand reaches a stable plateau after 4–5 more days in darkness,the proplastid structure becomes highly reduced. Forty minutesof light plus a one h dark period, or addition of glutamateor malate for 7 h does not change the proplastid structure significantlyeven though PT-Pchl(ide) returns to the level found in growingcells. Upon prolonged incubation in darkness after light treatment(72 h) an expanded proplastid containing prothylakoids, prolamellarbodies and membrane whorls with mitochondria in close associationis seen; most of the cellular paramylum is lost during thisperiod leaving cavities in the cytoplasm. Without light, prolongedincubation in darkness (72 h) with malate leads to accumulationof cellular paramylum but no change in proplastid structurewhile prolonged treatment with glutamate (72 h) allows the formationof a few prothylakoids but no prolamellar bodies. ¹Supported by Grants GM 14595 from the National Institutes ofHealth. ²Permanent address: Department of Microbiology, Tokyo MedicalCollege, 6-1-1 Shinjuku, Tokyo 160, Japan. ³Abraham and Etta Goodman Professor of Biology. (Received July 23, 1983; Accepted September 22, 1983)     

Carotenoid Synthesis in Leaves of Etiolated Wheat Seedlings after Varying Light and Dark Treatments

A short impulse of red light has a varying effect on carotenoid synthesis in dark-grown wheat seedlings. Except for β-carotene, which remains unchanged in the dark (it increases in continuous light), the carotenoid synthesis shows the same tendency as during constant irradiation. Thus, lutein and neoxanthin slowly increases, while violaxanthin decreases. During a period of constant light following various periods of darkness after the short impulse of light, the pigment changes correspond to those occurring in the dark, but are much more pronounced. The changes are discussed on the basis of phytochrome action.