Involvement of photobleaching and inhibition of protochlorophyll(ide) accumulation in tentoxin effects on greening of mung bean seedlings

Several types of evidence indicate that tentoxin-caused reduction of chlorophyll accumulation in greening primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] is due to both photobleaching and decreased protochlorophyll(ide) synthesis. Greening was greater under dim (2.5 μmol m_-2 s_-1) far-red or white light than under bright (180 to 200 μmol m_-2 s_-1) white light in tentoxin-treated tissues, whereas there was a positive correlation between fluence rate and greening in control tissues. Under continuous white light (100 μmol m_-2 s_-1) chorophyll(ide) accumulation was slower in tentoxin-treated than in control tissues. This was caused by greater photobleaching of newly formed chlorophyll(ide), as well as by decreased protochlorophyll(ide) synthesis. Photobleaching did not affect protochlorophyll(ide) synthesis in control or tentoxin-treated tissues. Chlorophyll(ide) was less stable in tentoxin-treated than in control tissues during a 24 h period of darkness. Plastids of tentoxin-treated tissues had all of the chlorophyll-proteins of control plants. Etioplasts of tentoxin-treated plants contained normal galactolipid contents, but galactolipids in these plants were greatly reduced in white light. Reduced chlorophyll accumulation caused by tentoxin is apparently the result of both photodestruction and of reduced synthesis of chlorophyll.     

Development and ageing of etioplast structures in dark grown leaves ofAvena sativa (L.)

Summary Etioplasts of dark grown plants contain a large paracrystalline prolamellar body (PLB) and, attached to this there are prothylakoid membranes (PTs).PLB-tubules inAvena are composed mainly of two saponins and include only a low percentage of other lipids, protochlorophyll(ide) and proteins.Following the development of etioplasts in darkness from the very beginning until plants loose turgescence one can observe marked changes in ultrastructure. In the early stage of development predominantly PTs are seen in small etioplasts. Wide-type PLBs are small. After eight days there is a well developed stage with the well-known big and highly crystalline PLBs, which are connected to many long PT-membranes. After 13 days the PLBs are not significantly changed, while number and length of PTs are strongly reduced.These morphological observations are quantified by measurements of PLB-area and PT-length per plastid section. Saponin content as a marker for PLB-tubules and protochlorophyll(ide)-content as a marker for PT-membranes were measured. Both methods of determination show in good agreement a peak of development for PTs around day 6–7, and for PLBs around 9–10. Beginning senescence affects PT-membranes and PChl(ide) strongly, while saponins resp. PLBs persist better. These results are presented in view of thylakoid formation during greening, starting from the different etioplast stages.Abbreviations Chl(ide) chlorophyll(ide) - EM electron-microscope - PChl(ide) protochlorophyll(ide) - PLB prolamellar body - PT prothylakoid - TLC Thin layer chromatography A preliminary report has been presented at the V. Intern. Congress on Photosynthesis at Chalkidiki, Greece 1980.     

Plastid differentiation and chlorophyll biosynthesis in different leaf layers of white cabbage (Brassica oleracea cv. capitata)

The contents of protochlorophyllide, protochlorophyll and chlorophyll together with the native arrangements of the pigments and the plastid ultrastructure were studied in different leaf layers of white cabbage (Brassica oleracea cv. capitata) using absorption, 77 K fluorescence spectroscopy and transmission electron microscopy. The developmental stage of the leaves was determined using the differentiation of the stoma complexes as seen by scanning electron microscopy and light microscopy. The pigment content showed a gradual decrease from the outer leaf layer towards the central leaves. The innermost leaves were in a primordial stage in many aspects; they were large but had typical proplastids with few simple inner membranes, and contained protochlorophyllide and its esters in a 2 : 1 ratio and no chlorophyll. Short‐wavelength, not flash‐photoactive protochlorophyllide and/or protochlorophyll forms emitting at 629 and 636 nm were dominant in the innermost leaves. These leaves also had small amounts of the 644 and 654 nm emitting, flash‐photoactive protochlorophyllide forms. Rarely prolamellar bodies were observed in this layer. The outermost leaves had the usual characteristics of fully developed green leaves. The intermediary layers contained chlorophyll a and chlorophyll b besides the protochlorophyll(ide) pigments and had various intermediary developmental stages. Spectroscopically two types of intermediary leaves could be distinguished: one with only a 680 nm emitting chlorophyll a form and a second with bands at 685, 695 and 730 nm, corresponding to chlorophyll–protein complexes of green leaves. In these leaves, a large variety of chloroplasts were found. The data of this work show that etioplasts, etio‐chloroplasts or chloro‐etioplasts as well as etiolated leaves do exist in the nature and not only under laboratory conditions. The specificity of cabbage leaves compared with those of dark‐grown seedlings is the retained primordial or intermediary developmental stage of leaves in the inner layers for very long (even for a few month) period. This opens new developmental routes leading to formation of specially developed plastids in the various cabbage leaf layers. The study of these plastids provided new information for a better understanding of the plastid differentiation and the greening process .     

Tentoxin effects on infrastructure and greening of ivyleaf morningglory (Ipomoea hederacea var. hederacea) cotyledons

The effects of 20 μM tentoxin on mesophyll chloroplast ultra-structural development, chlorophyll organization and accumulation, and pigment transformations in cotyledons of dark-grown, 4-day-old ivyleaf morningglory [Ipomoea hederacea (L.) Jacq. var. hederacea]were monitored. After 6 h of white light (200 μEm^?2T.s^?1), many plastids of tentoxin-treated tissues contained prolamellar bodies or inconsistent internal membrane orientation in contrast to the uniform internal membrane orientation and absence of prolamellar bodies in controls. Grana stacking did not progress beyond three to four disc loculi in tentoxin-treatments, and fret membranes were usually discontinuous and reduced. Cylindrical or cupped grana appeared in many chloroplasts after 3 days of light, while other chloroplasts in which disruption was more pronounced had few grana except for remnants, but usually did possess vesicles or structures resembling prolamellar bodies. Tentoxin had no apparent effect on stroma density or plastoglobuli size and number. No starch grains appeared in any of the tentoxin treatments, whereas they appeared after 24 h in controls. Initial protochlorophyllide content and its photoconversion to chlorophyllide and subsequent Shibata shift were not affected by tentoxin. Chlorophyll accumulation rates in tentoxin-treated cotyledons were about 10% of control rates during the first 24 h of greening and about 20% of controls from 48 to 72 h of greening. Chlorophyll alb ratio and PSU size (total Chl/P700) were not significantly affected by tentoxin.     

The Effects of Tentoxin on Chlorophyll Synthesis and Plastid Structure in Cucumber and Cabbage

To determine if chlorosis caused by tentoxin, a toxin produced by Alternaria tenuis Nees., is due to interference with chlorophyll synthesis directly or to disruption of normal chloroplast development, the effects of the toxin on these processes in cucumber (Cucumis sativus L.) and cabbage (Brassica oleracea L., var. capitata) were studied. Cucumber cotyledons are highly sensitive to the toxin but exhibited no interference with the conversion of protochlorophyll(ide) to chlorophyll(ide) or with the general time course pattern of chlorophyll synthesis, although there was a 90% reduction in chlorophyll concentration. In cabbage, which shows no chlorosis in the presence of the toxin, there was a slight stimulation of chlorophyll synthesis in the presence of the toxin. Electron microscopy revealed that in cucumber, toxin treatment interferes with development of prolamellar bodies and lamellae, and results in deformed plastids. No such effects were noted in toxin-treated cabbage tissues. Plastids in toxin-treated cotyledons of both cucumber and cabbage contained more starch than plastids in nontreated tissues. It was concluded that tentoxin acts through disruption of normal plastid development, rather than through direct interference with chlorophyll synthesis.     

Formation of short-wavelength chlorophyll(ide) after brief irradiation is correlated with the occurrence of protochlorophyll(ide)636–642 in dark-grown epi- and hypocotyls of bean (Phaseolus vulgaris)

Chlorophyll formation capacity along the seedling of bean ( Phaseolus vulgaris L. cv. Brede zonder draad) was investigated. After 7 days of irradiation a gradient was formed, where the primary leaf contained ca 300 times more chlorophyll per gram fresh weight than the lower hypocotyl section and ca 20 times more than the epicotyl. Similar chlorophyll gradients but at lower levels were seen when the seedlings were first placed in darkness for 7 days and then irradiated for 1, 2 or 7 days. Ultrastructural investigation of seedlings grown for 7 days in darkness and then irradiated for 24 h revealed a more developed inner membrane system with grana stacks in plastids of cells in the uppermost hypocotyl section compared to plastids of cells in lower hypocoty] sections. The higher up on the seedling the more the ratio increased of protochlorophyll(ide) emitting at 657 nm to short-wavelength protochlorophyll(ide). After flash irradiation of the different sections, fluorescence emission spectra with maxima at 680 and 690 nm, respectively, were observed, indicating the formation of short- and long wavelength chlorophyll(ide) forms. The lower the ratio of protochlorophyll(ide) emitting at 657 nm to the short-wavelength protochlorophyll(ide), the less long-wavelength chlorophyll(ide) was formed after irradiation. However, after continuous irradiation long-wavelength chlorophyll(ide) was formed. In dark grown roots, where only short-wavelength protochlorophyll forms were present, it was not possible to transform protochlorophyll to chlorophyll by flash irradiation. Possible explanations for this phenomenon are discussed.     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Oak seedlings grown in different light qualities

Oak seedlings (Quercus robur L.) were germinated in darkness for 3 weeks and then given continuous long wavelength far-red light (LFR; wavelengths longer than 700 nm). A control group of seedlings was kept in darkness. After 2 additional weeks the chlorophyll formation ability in red light was examined in the different seedlings. The stability of the protochlorophyll(ide) and chlorophyll(ide) forms to high intensity red irradiation was also measured. Oak seedlings grown in darkness accumulated protochlorophyll(ide) (6 μg per g fresh matter). Absorption spectra and fluorescence spectra indicated the presence of more protochlorophyll(ide)_628–632 than protochlorophyllide_650–657. The level of protochlorophyll(ide) was higher in leaves of plants cultivated in LFR light (13 μg per g fresh matter) than in leaves of dark grown plants. 12% of the protochlorophyll(ide) was esterified in both cases. The level of protochlorophyll(ide)_628–632 in LFR grown oaks varied with the age of the leaves, being higher in the older (basal) leaves, but also in the very youngest (top-most) leaves. The ability of the leaves to form photostable chlorophyll in red light showed a similar age dependence, being low in rather young and in older leaves. A low ability to form photostable chlorophyll thus appears to be correlated with a high content of protochlorophyll(ide)_628–632. Upon irradiation only the protochlorophyllide_650–657 was transformed to chlorophyllide. After this phototransformation the chlorophyllide peak at 684 nm shifted to 671 nm within about 30 min in darkness. This shift took place without any accompanying change in photostability of the chlorophyll(ide). Upon irradiation with strong red light a similar shift took place within one minute. This indicates that the chlorophyllide after phototransformation was rather loosely bound to the photoreducing enzyme. The development towards photostable chlorophyll forms consists of three phases and is discussed.     

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.     

An ultrastructural study of maize leaf etioplasts throughout their entire life-cycle

Summary The entire life-cycle of maize leaf etioplasts has been followed. Prolamellar bodies with different types of tubular membrane arrangement can be found in the juvenile stages of the organelles, while in mature etioplasts nearly all the prolamellar bodies exhibit an hexagonal ring arrangement, which, by optical diffraction, appears to be the most regular and compact possible.The prothylakoid membranes also undergo changes during organelle differentiation, and their different organization and arrangement produce a clear dimorphism between the etioplasts of mesophyll and bundle sheath cells.In senescent etioplasts the prothylakoids are more affected, while the prolamellar bodies appear rather stable, also in situations where protochlorophyll(ide) content is very low. The formation of clusters of osmiophilic globules is coupled with the breakdown of the etioplast membranes.     

Developmental Physiology of Bean Leaf Plastids III. Tube Transformation and Protochlorophyll (ide) Photoconversion by a Flash Irradiation

A light flash of about 1 millisecond duration elicits tube transformation in paracrystalline prolamellar bodies as well as maximal protochlorophyll(ide) photoconversion in etiolated bean leaves (Phaseolus vulgaris L.). These findings support a more detailed hypothesis on the linkage between tube transformation and protochlorophyll(ide) photoconversion than has been offered previously.     

Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)     

Events Surrounding the Early Development of Euglena Chloroplasts: I. Induction by Preillumination 1

Preillumination, followed by a dark period prior to exposure of dark-grown nondividing cells of Euglena gracilis var. bacillaris to normal lighting conditions for chloroplast development, results in potentiation, or abolishment of the usual lag in chlorophyll accumulation. The degree of potentiation is a function of the length of the preillumination period, the intensity of preilluminating light, and the length of the dark period interposed before re-exposure to continuous light for development. The optimal conditions are found to be: 90 minutes of preillumination with white light at an intensity greater than 30 microwatts per square centimeter (14 foot candles) followed by a dark period of at least 12 hours. Reciprocity is not found between duration and intensity of preilluminating light. Preillumination with blue light and red light was found to be the most effective in promoting potentiation, and the ratio of effectiveness of blue to green to red is consistent with protochlorophyll-(ide) being the photoreceptor. Although red light is effective, there is no reversal by far red light, and these facts, taken together with the effectiveness of blue light, suggest that the phytochrome system is not involved. The amount of chlorophyll formed at the end of preillumination is proportional to the resulting potentiation, suggesting that the amount of protochlorophyll(ide) removed or chlorophyll(ide) formed regulates this phenomenon. Potentiated and nonpotentiated cells show comparable rates of protochlorophyll(ide) resynthesis, suggesting that this is not the limiting factor in nonpotentiated cells. Although light is required for protochlorophyll(ide) conversion in chlorophyll synthesis, a brief preillumination seems also to initiate the production of components in the subsequent dark period which, in nonpotentiated cells, are ordinarily synthesized during the lag period under continuous illumination. These components are necessary to sustain maximal rates of subsequent chlorophyll accumulation.     

The Correlated Appearance of Prolamellar Bodies, Protochlorophyll(ide) Species, and the Shibata Shift during Development of Bean Etioplasts in the Dark

The structure and physiology of the etioplast was investigated in developing primary leaves of 3- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Increase in total protochlorophyll(ide) content followed that of leaf fresh weight. In 3- to 4-day-old bean leaves more than 50% of the protochlorophyll(ide) is in the form of protochlorophyll(ide) 628, which is nontransformable by light. Most of the transformable pigment is protochlorophyll(ide) 635, with smaller amounts of protochlorophyll(ide) 650. During leaf development from the 3rd to the 7th day phototransformable protochlorophyll(ide) with an absorbance maximum at 650 nm accumulates faster than nontransformable protochlorophyll(ide) or protochlorophyll(ide) 635. This increase in protochlorophyll(ide) 650 is correlated with the formation and enlargement of prolamellar bodies.     

Studies on a Maize Mutant Sensitive to Low Temperature II. Chloroplast Structure, Development, and Physiology

In the Zea mays L. mutant M11 grown in the dark at 15°, the ultrastructure of the etioplast is abnormal. The pigment content of the etioplasts is reduced but the in vivo absorption characteristics suggest that the normal protochlorophyll (ide)-holochrome is present. The lowered synthetic ability of the etioplasts is not primarily due to a reduced complement of plastid ribosomes. The plastids of mutant M11 grown in the light at 15° contain little pigment, are markedly deficient in ribosomes and their ultrastructure is abnormal. In mutant M11 grown at 15°, an extreme sensitivity of the plastid membranes to light was observed.     

Light-induced structural changes during incubation of isolated maize etioplasts

Summary Etioplasts were isolated from leaves of 9-day-old etiolated maize (Zea mays L.) seedlings and incubated in a relatively simple medium in light and in the dark. During the first 5 h no changes occurred in the fine structure of the isolated etioplasts in the dark. In light the size of the prolamellar bodies decreased and significantly more plastid sections without prolamellar bodies were counted. The total length of the thylakoids per plastid section increased, but there was no evidence for bi- and polythylakoid formation. It is concluded that light induces the structural transformation of the prolamellar body membranes into primary thylakoids also in isolated etioplasts.     

Structure of Protochlorophyll-containing Plastids in the Inner Seed Coat of Pumpkin Seeds (Cucurbita pepo)

The inner seed coat of seeds of Cucurbita pepo L. cv. Ohlsens Enke Köks was used to study the development of protochlorophyll-containing plastids with an abnormal ultrastructural composition. The pumpkins were harvested at different stages during fruit development and they thus contained seeds with different developmental stages. The dry weight of seeds of the developmental stages used varied from 0.04 g to 0.3 g. Such a series of seeds with decreasing water content indicating increasing maturity contained different amounts of protochlorophyll, from 0.20 μg/g fresh weight to 500 μg/g fresh weight. The ultrastructure of the protochlorophyll containing plastids changed greatly during development. In young seeds with a low content of protochlorophyll, regular prolamellar bodies were found and starch grains filled most of the plastids. During development the starch content decreased and the prolamellar bodies increased in size and lost their regularity. During maturation the plastids accumulated plastoglobuli, probably containing protochlorophyll, and finally the internal structure of the prolamellar body tubular complex was lost.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.     

Transformation of plastids in soil‐shaded lowermost hypocotyl segments of bean (Phaseolus vulgaris) during a 60‐day cultivation period

The maintenance but substantial transformation of plastids was found in lowermost hypocotyl segments of soil‐grown bean plants (Phaseolus vulgaris cv. Magnum) during a 60‐day cultivation period. Although the plants were grown under natural light–dark cycles, this hypocotyl segment was under full coverage of the soil in 5–7 cm depth, thus it was never exposed to light. The 4‐day‐old plants were fully etiolated: amyloplasts, occasionally prolamellar bodies, protochlorophyllide (Pchlide) and protochlorophyll (Pchl) were found in the hypocotyls of these young seedlings. The 633 and 654 nm bands in the 77 K fluorescence emission spectra indicated the presence of Pchlide and Pchl pigments. During aging, both the Pchlide and Pchl contents increased, however, the Pchl to Pchlide ratio gradually increased. In parallel, the contribution of the 654 nm form decreased and in the spectra of the 60‐day‐old samples, the main band shifted to 631 nm, and a new form appeared with an emission maximum at 641 nm. The photoactivity had been lost; bleaching took place at continuous illumination. The inner membranes of the plastids disappeared, the amount of starch storing amyloplasts decreased. These data may indicate the general importance of plastids for plant cell metabolism, which can be the reason for their maintenance. Also the general heterogeneity of plastid forms can be concluded: in tissues not exposed to light, Pchl accumulating plastids develop and are maintained even for a long period.     

Protochlorophyll(ide) accumulation and degradation in the dark and photoconversion to chlorophyll in the light in pigment mutant C-2A' of Scenedesmus obliquus

Pigment mutant C-2A' of Scenedesmus obliquus accumulates only traces of chlorophyll, when grown heterotrophically in the dark. Immediately upon transfer of cells into fresh medium protochlorophyllide and protochlorophyll are formed, which accumulate to their maximum concentrations within 8 to 12 h. Subsequently, this protochlorophyll(ide) is degraded in the dark, but not transformed into chlorophyll. After 6–8 days of dark growth no protochlorophyll(ide) can be detected any more. The protochlorophyll(ide) pool of cultures, which contain reduced concentrations, can be reestablished either by addition of glucose or illumination with blue light; both increase the rate of respiration.
By low temperature spectroscopy in vivo and by absorption and fluorescence emission spectroscopy of pigment extracts it is shown that the protochlorophyllide accumulated in freshly inoculated cultures can be converted to chlorophyll in light.
From the action spectrum for chlorophyll formation after addition of glucose it can be seen that protochlorophyllide 636 and 649 are present and are photoconvertible in this mutant.