Pigment-protein complexes of illuminated etiolated leaves

Photoconversion of protochlorophyllide in etiolated leaves of Avena sativa L., var. Pennal or Peniarth and Phaseolus vulgare L., var. `The Prince' results in the sequential appearance of spectrally distinct chlorophyllide complexes (Chlide 678, 684, and 672). This paper reports on the generation of similar forms in vitro, under controlled conditions, using well characterized etioplast membranes enriched in the enzyme protochlorophyllide reductase. Excess NADP<sup>+</sup> and NADPH stabilize complexes related to Chlide 678 and Chlide 684, respectively, whereas addition of exogenous Pchlide induces formation of a species related to Chlide 672. Evidence is provided to support the suggestion that Chlide 678 and Chlide 684 represent ternary complexes of the enzyme protochlorophyllide reductase, with Chlide and either NADP<sup>+</sup> (Chlide 678) or NADPH (Chlide 684). Chlide 672 is seen as `free' pigment dissociated from the enzyme. The role of Pchlide in this dissociation, observed spectroscopically as the `Shibata shift,' is discussed.     

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2° the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2° was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2° varied 3-fold among the plants treated. The reaction was not first order. At 25° the reaction followed at 690 nm was complete in 20 to 60 sec. Q₁₀'s varied from 2.8 to 3.7 between 2° and 25°. Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.     

Light-regulated pigment interconversion in pheophytin/chlorophyll-containing complexes formed during plant leaves greening

Upon illumination of etiolated maize leaves the photoconversion of protochlorophyllide Pchlide 655/650 into chlorophyllide Chlide 684/676 was observed. It was shown that chlorophyllide Chlide 684/676 in the dark is transformed into pheophytin Pheo 679/675 and chlorophyll Chl 671/668 by means of two parallel reactions, occurring at room temperature: Chlide 684/676. The formed pheophytin Pheo 679/675 was unstable and in the dark was transformed into chlorophyll Chl 671/668 in a few seconds: Pheo 679/675 Chl 671/668. The last reaction is reversed by the light: Chl/668 Pheo 679/675. Thus, on the whole in the greening etiolated leaves this process occurs according to the following scheme:The observed light-regulated interconversion of Mg-containing and Mg-free chlorophyll analogs is activated by ATP and inhibited by AMP.Abbreviations Chl- chlorophyll - Chlide- chlorophyllide - Pchlide- protochlorophyllide - Pheo- pheophytin - PS II RC- Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to the maximum position of the low-temperature fluorescence band (nm), the second number to the maximum position of the longwave absorption band     

Biosynthesis of non-fluorescent chlorophyll of Photosystem II core in greening plant leaves. Effect of etiolated plants growing under heat shock conditions (38 °C)

Preliminary dark incubation of etiolated pea and maize plants at 38 °C allowed to observe a new dark reaction of Chl biosynthesis occuring after photoconversion of protochlorophyllide Pchld 655/650 into chlorophyllide Chld 684/676. This reaction was accompanied by chlorophyllide esterification and by the bathochromic shift of pigment spectra: Chld 684/676 Chl 688/680. After completion of the reaction, a rapid (20–30 s at 26 °C) quenching of Chl 688/680 low-temperature fluorescence was observed. The reaction Chld 684/676 Chl 688/680 was inhibited under anaerobic conditions as well as in the presence of KCN; the reaction accompanied by Chl fluorescence quenching was inhibited in the leaves of pea mutants with impaired function of Photosystem II reaction centers. The spectra position of newly formed Chl, effects of Chl fluorescence quenching allowed to assume that the new dark reaction is responsible for biosynthesis of P–680, the key pigment of Photosystem II reaction centres.     

Esterification and Spectral Shifts of Chlorophyll(ide) in Wildtype and Mutant Seedlings Developed in Darkness

Spectral changes and esterification (presumably with phytol) of newly formed chlorophyllide a in dark-grown leaves of wildtype bean (Phaseolus vulgaris) and barley (Hordeum vulgare) and a number of chloroplast mutants in barley, were studied by spectrofluorimetry on leaves and on solvent extracts. The shift of the fluorescence emission maximum from 692–694 to 678 nm (excitation shift: 682–684 to 672 nm) and esterification of chlorophyllide a have a similar time course, and both processes are temperature dependent in a similar manner. After completion of the spectral shift and esterification, the fluorescence efficiency of chlorophyll a increases with a subsequent reaccumulation of protochlorophyllide. In leaves of mutants where the shift of fluorescence from 692 to 678 nm is lacking, esterification and the subsequent processes are also blocked. In leaves of mutants with a rapid shift of the fluorescence from 692 to 678 nm, or with direct photoconversion to chlorophyllide a with the fluorescence at 678 nm, esterification is also rapid. The results are interpreted as a sequence of molecular events involving a conformational relaxation of the chlorophyllide holochrome and a translocation of chlorophyll a to reaction centers of the photosystems.     

A comparative study of the terminal stages of chlorophyll biosynthesis before and after heavy water (D2O) introduction into greening plant leaves

By spectral methods, the final stages of chlorophyll formation from protochlorophyllide proceeding in intact greening maize leaves were studied before and after the introduction of heavy water (D₂O) into etiolated leaves. Three effects of D₂O introduction were observed: 1) a complete inhibition of the reaction pathway leading to pheophytin biosynthesis and formation of pheophytin/chlorophyll-containing complexes (presumably, direct precursors of Photosystem II reaction centers): 2) 5-fold inhibition of the reaction of the Shibata shift ; 3) appearance of a new dark reaction of the primary chlorophyllide native form Chld 684/676 'Chld 690/680'. It was shown that the intermediate Chld 684/676 presents the point of a triple branching of chlorophyllide transformation; activities of these three parallel pathways of Chld 684/676 transformation can be regulated by light intensity as well as by temperature.     

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.     

Fast phototransformation of the 636 nm-emitting protochlorophyllide form in epicotyls of dark-grown pea (Pisum sativum)

The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m⁻² s⁻¹) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m⁻² s⁻¹) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.     

The Relation Between the Phytylation and the 682 → 672 nin Shift in vivo of Chlorophyll a

The rate of phytylation and the rate of the in vivo Chl₆₈₂→ Chl₆₇₂ nm shift of the newly formed chlorophyllide were examined after a brief illumination of dark-grown Phaseolus vulgaris leaves at different stages of development. Both processes were found to be age dependent but independent of one another. The Chl₆₈₂→ Chl₆₇₂ nm shift process precedes that of phytylation. In addition, there is a linear relationship between tissue age and the delay time between the two processes, indicating that they are not identical, although they are almost parallel. There was no evidence obtained of a far-red light effect on any of the two processes. Experiments done with freeze-dried etiolated plant tissue showed the presence of PChl₆₅₀ and PChl₆₃₇ forms, which by illumination form Chl₆₇₈. If a trace of water is added to the etiolated freeze-dried tissue before illumination the PChl₆₅₀ and PChl₆₃₇ are transformed to the non-phototransformable PCh₆₅₀ form. Moreover, no shift of the Chl₆₇₈ form is observed, unless a trace of water is added to the freeze-dried tissue. These results are explained by a mechanism of structural rearrangement in the chloroplast after protochlorophyllide phototransformation leading into monomeric chlorophyllide units, which can then be phytylized. This explanation is substantiated by the results of experiments done with isolated protochlorophyllide-holochrome: addition of 4M urea before or after illumination to the protochlorophyllide-holochrome solution showed a PChl₆₃₆→ PChl₆₂₉ and Chl₆₇₆→ Chl₆₆₈ shift respectively.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

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.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.     

Breakdown of photosynthetic pigments and lipids in spinach leaves with ozone fumigation: Role of active oxygens

Dark-grown wheat leaves ( Triticum L. cv. Starke II Weibull) were illuminated repeatedly with light flashes giving partial phototransformation of protochlorophyllide to chlorophyllide. After short flashes (e.g. 15 ms red light, 250 W m⁻²), transforming only a minor part of the protochlorophyllide present, the first more stable chlorophyll(ide) measured ca 15 s after the phototransformation had its absorption maximum in the red around 672 nm. It stayed there during the following 30 min in darkness. After longer flashes (e.g. 125 ms), transforming a larger portion of the protochlorophyllide, the chlorophyll(ide) formed had its maximum absorption more towards 684 nm and shifted to 672 nm during a subsequent period in darkness. Thus, in this case a Shibata shift took place.
The conditions which produce the "stable" 672 nm form, without a Shibata shift, are discussed. The presence of large amounts of non-transformed protochlorophyllide remaining after the phototransformation seems to be important. Under such conditions it is possible that the Shibata shift is completed within a very short time.
Also the possible existence of two kinds of phototransformable protochlorophyllide is discussed. According to this idea one of the two protochlorophyllide forms produces a chlorophyllide absorbing at 672 nm shortly after phototransformation without having passed a Shibata shift. The other protochlorophyllide form photo-transforms to a chlorophyllide which proceeds through the Shibata shift.     

Photosynthesis in the Higher Plant, Vicia faba: III. Serine, a Precursor of the Tricarboxylic Acid Cycle

Evidence is presented to support the hypothesis that serine, rather than 3-phosphoglycerate of the Calvin cycle, is a precursor of the tricarboxylic acid cycle during photosynthesis by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C₁ > C₂ > C₃ isotope distribution for that metabolite following the fixation of ¹⁴CO₂. This labeling pattern, while incompatible with an origin either in the Calvin cycle or the glycolate pathway, satisfies a critical criterion for the 3-carbon precursor of the anomalously labeled organic acids. The predominant carboxyl carbon atom labeling of serine reflects either a mixing of two pools of that metabolite, ie., C₁ = C₂ > C₃ and C₁ > C₂ = C₃, or a higher order of complexity in its synthesis. An anomalous C₁ = C₂ > C₃ < C₄ distribution for aspartate, however, suggests an origin by the carboxylation of a 3-carbon intermediate related to serine which has a C₁ = C₂ > C₃ distribution. The latter distribution has been proposed for the serine intermediate of the postulated formate pathway. This pathway is described by the generalized metabolic sequence: CO₂ → formate → serine → organic acids. Corresponding carbon atom distributions for citrate (C₁ > C₂), aspartate (C₂ > C₃), and serine (C₂ > C₃) belie a precursor-product relationship with alanine (C₂ = C₃), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate.     

Heparin: New Biochemical and Medical Aspects : Proceedings of the Symposium of the Deutsche Gesellschaft für Klinische Chemie,Titisee, Breisgau,German Federal Republic,June 29th - July 1st, 1981 Edited by I. Witt Walter de Gruyter; Berlin,New York, 1983 372 pages. DM 155.00

Using a phosphoroscopic attachment to the dichrograph, light-induced circular dichroism spectra have been measured for chlorophyll-protein complexes of Photosystem I. Minor components at 672, 678 and 685 nm are observed in these spectra in addition to the components of dimer splitting of the P700 Q_y transition at 691 and 698 nm. The minor components are due to the Chl672, Chl₆₇₈ and Chl₆₈₅ forms of antenna chlorophylls, the optical activity of which is changed 2–4% as a result of P700 oxidation. It is suggested that P700 is not an isolated dimer but that it is included in a local complex comprising 8–10 chlorophyll molecules with an exciton level splitting value of 120–140 cm^?1.     

The Shibata Shift and the Transformation of Etioplasts to Chloroplasts in Wheat with Clomazone (FMC 57020) and Amiprophos-Methyl (Tokunol M)

The Shibata shift is a change in the absorption maximum of chlorophyllide from 684 to 672 nanometers that occurs within approximately 0.5 hour of phototransformation of protochlorophyllide to chlorophyllide. Two compounds, clomazone and amiprophos-methyl, which previously have been shown to inhibit the Shibata shift in vivo, were used to look for correlations between the Shibata shift and other processes that occur during etioplast to chloroplast transformation. Leaf sections from 6-day-old etiolated wheat seedlings (Triticum aestivum L. cv Walde) were treated with 0.5 millimolar clomazone or 0.1 millimolar amiprophos-methyl in darkness. In addition to the Shibata shift, the esterification of chlorophyllide to chlorophyll and the relocation of protochlorophyllide reductase from the prolamellar bodies to the developing thylakoids were inhibited by these treatments. Prolamellar body transformation did not appear to be affected by amiprophos-methyl and was only slightly affected by clomazone. The results indicate that: (a) there is a strong correlation between the occurrence of the Shibata shift and esterification activity; (b) transformation of the prolamellar bodies does not depend on the Shibata shift; and (c) the occurrence of the Shibata shift may be a prerequisite to the relocation of protochlorophyllide reductase from prolamellar bodies to thylakoids.     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of ?15 – ?80°C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.