Control of chloroplast formation by light.

Light controls the formation of plastid ultrastructure and the synthesis of chlorophyll, plastid membrane constituents and Calvin cycle enzymes. A respective light-mediated regulation of the genetic apparatus in the nucleus and the plastid compartment has been reported. Three photoreactions are involved in the regulation: (1) the protochlorophyll (ide) leads to chlorophyll (ide) a photoconversion, (2) the formation of physiologically active phytochrome and (3) light absorption by a blue light receptor (cryptochrome). The chloroplast formation in higer plants is chiefly controlled by active phytochrome, while in lower plants cryptochrome is the prevailing regulatory factor.

[The action of phytochrome and actinomycin D on chlorophyll a formation in mustard seedlings (Sinapis alba L.)]. / Die Wirkung von Phytochrom und Actinomycin D auf die Chlorophyll a-Synthese von Senfkeimlingen (Sinapis alba L.).

In the mustard seedling chlorophyll a synthesis under white light is enhanced by a pretreatment with far-red which maintains a low but virtually stationary concentration of active phytochrome (=P730) (Fig. 1) during the period of irradiation (4 hours). - On the other hand chlorophyll a synthesis is inhibited or delayed by relatively low concentrations of Actinomycin D(=Act) (Fig. 2)The inhibitory action of Act (on a percent basis) is exactly the same with and without a far-red pre-irradiation (Fig. 3). Act in relatively low doses (5 or 10 µg/ml) greatly extends the lag-phase of chlorophyll synthesis; however, these doses do not influence the effect of the far-red pretreatment on the rate of chlorophyll synthesis when it finally takes place (Fig.4,5,6). The data presented in this paper indicate that Act does not inhibit protochlorophyll synthesis as such; we have rather to conclude that Act inhibits the de novo synthesis of some specific structural proteins which are prerequisites of chlorophyll accumulation and maintenance in the plastids (Table 1). Synthesis of these structural proteins seems to be under the control of phytochrome too.It is concluded that those genes which are already in function are relatively resistant to Act (e. g. those genes which are needed for protochlorophyll synthesis) whereas potentially active genes (e. g. those which code some specific structural proteins of the plastids) are very sensitive to Act. -A similar conclusion has been reached in an earlier paper in connection with phytochrome-induced antocyanin synthesis (LANGE and MOHR, 1965). Our argumentation is further supported by SCHOPFER's data on control of ascorbate synthesis in the mustard seedling by phytochrome and Act (SCHOPFER, 1966).

Control of chlorophyll synthesis by phytochrome : III. Does phytochrome regulate the chlorophyllide esterification in mustard seedlings?

The rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)→chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (Pfr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that Pfr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by Pfr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55-58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of Pfr.

Control of chlorophyll synthesis by phytochrome : I. The effect of phytochrome on the formation of 5-aminolevulinate in mustard seedlings.

Treatment of mustard (Sinapis alba L.) seedlings with levulinate leads to the inhibition of chlorophyll synthesis and causes the accumulation of 5-aminolevulinate which is only formed in light. A stoichiometric relationship exists between the extent of inhibition of chlorophyll synthesis and 5-aminolevulinate accumulation. The formation of 5-aminolevulinate in continuous white light is increased by pre-irradiation. The effect of the preirradiation can be fully attributed to phytochrome. Under various light conditions the rate of 5-aminolevulinate formation in levulinate-treated seedlings is similar to the rate of chlorophyll accumulation in seedlings not treated with levulinate. This result supports the hypothesis that the phytochrome-controlled chlorophyll accumulation is regulated at the level of the formation of 5-aminolevulinate.

Control of chlorophyll synthesis by phytochrome : II. The effect of phytochrome on aminolevulinate dehydratase in mustard seedlings.

The activity of aminolevulinate dehydratase in mustard (Sinapis alba L.) seedlings increases in continuous far-red light. The light effect can be attributed to phytochrome. The same was found for the accumulation of protochlorophyll(ide) if the seedlings were treated with 5-aminolevulinate. This result could indicate that a considerable portion of the aminolevulinate dehydratase is located in the plastids. No correlation exists between aminolevulinate dehydratase activity and the capacity of the mustard seedlings to form chlorophyll. In conclusion, the increase in enzyme activity is probably not involved in the phytochrome-mediated control of chlorophyll biosynthesis.

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris).

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm(-2)) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (Pfr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.

Coaction of three factors controlling chlorophyll and anthocyanin synthesis.

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, P fr) and an excision factor. It was found that the three factors operate additively provided that the P fr level is high enough. When the P fr level is below approximately 1 per cent (ϕλ<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low P fr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.

Correlation between 5-aminolaevulinate accumulation and protochlorophyll photoconversion.

A 1-min light pulse delivered to mustard seedlings (Sinapis alba L.) 60 h after sowing initiates the release of cotyledonary 5-aminolaevulinate (ALA) accumulation which continues for at least 2 h in the dark. Phytochrome (P fr) increases the rate of ALA accumulation after a 24-h red light pretreatment but is not the trigger for this release. It is shown that the rate of ALA accumulation varies with the wave-length and fluence rate of the 1-min light pulse and can be predicted from the degree of protochlorophyll-(ide) photoconversion. There is a linear correlation between the rate of ALA accumulation and the degree of protochlorophyll(ide) (PChl)→chlorophyll(ide) a (Chl a) photoconversion in etiolated seedlings. In seedlings pretreated with red light this correlation is non-linear and the rate increases more rapidly with increasing degrees of PChl→Chl a photoconversion. It is suggested that there may exist an interaction between P fr and PChl→Chl a photoconversion in controlling ALA accumulation.

The capacity of chlorophyll-a biosynthesis in the mustard seedling cotyledons as modulated by phytochrome and circadian rhythmicity.

Within the temporal pattern of "primary differentiation" the capacity of chlorophyll - a biosynthesis in the cotyledons ofSinapis alba L. seedlings is controlled by phytochrome (in continuous light) or by releasing the circadian rhythm either with lightdark cycles or by a light→dark transition. The sensor pigment for this process is phytochrome. It is very probable that in continuous light as well as under conditions under which the circadian rhythm plays the major part, the capacity of chlorophyll a biosynthesis is limited by the capacity of the biosynthetic step which produces 5-aminolaevulinate.

Timing of the initial action of phytochrome with regard to protochlorophyll synthesis in the mustard seedling.

Significant accumulation of photoconvertible protochlorophyll(ide) in the cotyledons of the mustard seedling takes place from 24 h after sowing onwards (25° C). The rate of accumulation in darkness is greatly increased by a pretreatment with red or far-red light. The strong effect of continuous red light, given from the time of sowing, remains fully reversible by a 756 nm-light pulse up to about 18 h after sowing. On the other hand, the effect of continuous far-red light which can be detected at 15 h after sowing is not influenced by a subsequent application of 756 nm-light pulses. An interpretation of the data requires the concept that continuous red light and continuous far-red light act from different sites. This conclusion is based on a comparison of the present data with the earlier published data on phytochromemediated anthocyanin synthesis in the mustard seedling cotyledons.

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons.

The etioplast¼chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.

Coaction of light and cytokinin in photomorphogenesis.

Intact mustard seedlings were treated with zeatin and photomorphogenetically active light in different ways: (1) hormone treatment preceding light treatment, (2) light treatment preceding hormone treatment, (3) hormone and light applied simultaneously. Under all experimental conditions the effect of the hormone treatment is multiplicative to the light effect with regard to the increase of cotyledon area. However, the hormone effect is additive to the light effect with regard to increases of the level of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and carotenoid contents. Anthocyanin synthesis is inhibited by exogenous zeatin whereby the concentration response curves are similar, irrespective of the extent of anthocyanin formation mediated by light. However, an interaction was found in the sense that the responsiveness toward zeatin is decreased somewhat by the action of phytochrome. Our results show that the responsiveness to light (via the far-red-absorbing form of phytochrome; P fr) is not changed by a preceding or simultaneous hormone treatment. Moreover, the responsiveness of the plant to exogenously applied zeatin is not affected - except in anthocyanin synthesis - by a preceding or simultaneous light treatment. We conclude from our results that the action of phytochrome on the developmental processes is not related to cytokinin levels.

Phytochrome-mediated delay of plastid senescence in mustard cotyledons: changes in pigment contents and ultrastructure.

Changes in pigment contents and ultrastructure have followed in cotyledons of mustard (Sinapis alba L.) seedlings during dark-mediated senescence. The seedlings were kept in white light for 7 d, treated with 5 min long wavelength far-red light and then kept in darkness up to 14 d after sowing. Under these conditions the chloroplasts remain stable for 2 d before a sequential plastidal disintegration commences. The data indicate a selective breakdown of the light-harvesting chlorophyll a/b protein. Phytochrome retards the differential loss of chlorophyll a, b and carotenoids and preserves the fine structure of chloroplasts.