SUMMARY Preparations of isolated chloroplasts show a reduction of photophosphorylation activity due to ageing even when stored in the dark at 0–4°C in a stabilizing medium. The rate of activity loss depends strongly upon the type of photophosphorylation. With non‐cyclic photophosphorylation (ferricyanide or NADP‐ferredoxin) reduction of activity is rapid immediately after isolation of chloroplasts, whereas it decreases to much slower rates after 3–4 hours. In the presence of PMS, which under our reaction conditions serves as electron acceptor for PS‐2 and electron donor for PS‐1, photophosphorylation activity is hardly affected by the increasing age of chloroplasts. However, upon addition of low concentrations of DCMU, reduction of this phosphorylation activity due to ageing shows the same kinetics as non‐cyclic photophosphorylation. Photophosphorylation of PS‐1 (reduced DCPIP‐MVDCMU) is hardly affected by the ageing of chloroplasts.

Action spectra of PS-I photophosphorylation show absorption bands or shoulders with peak locations around 640, 647-650, 660, 672-674, 680, 690, 701-703, 710-712 and 721 nm. Phosphorylation activity is reduced around 665-667 and 676 nm suggesting decreased absorption bands of pigments involved in the light absorption process of PS-2.

Abstract— Photophosphorylation action spectra were measured with the utmost precision to identify the different pigment forms involved in the light‐harvesting process for formation ofadenosine–5′‐triphosphate (ATP). With ferricyanide‐ions as electron acceptor freshly prepared thylakoid membranes yield a typical photosystem 2 (PS II) action spectrum peaking at 672 nm and with pronounced shoulders at 650 and 680 nm. Because ferricyanide‐ions can be reduced by both photosystems we suggest that due to the poor accessibility of these hydrophilic ions to the PS II acceptor site, the PS II electron flow is rate‐limiting. When the thylakoid membranes are aged, the maximum of the action spectrum shifts to 675 nm and bands in the spectral region690–725 nm are observed, which are generally attributed to photosystem 1 (PS I). Obviously, as a result of the aging, the rate‐limiting effect of PS II electron flow decreases and activity of both photosystems is observed. With nicotinamide adenine dinucleotide phosphate (NADP) and ferredoxin as electron acceptors also a typical PS II action spectrum is obtained peaking at 676 nm and with a sharp drop in activity at 695 nm. However, the spectrum shows a pronounced peak at706–707 nm, which is attributed to a pigment involved in the light‐harvesting for either of the two photosystems. Curve‐analysis of the action spectra suggests that four forms of Chi a exist at room temperature in the spectral region660–685 nm. However, a definite conclusion about the occurrence of different pigment forms will be drawn on the basis of combined results from this and the following publication. This is done to yield a conclusion that is statistically more reliable. ATP–adenosine‐5′‐triphosphate; BSA– bovine serum albumin; Chl–chlorophyll; DBMIB–2,5‐dibromo‐3‐methyl‐6‐isopropylbenzoquinone; DCMU–3‐(3,4)‐dichlorophenyl‐1,1‐dimethyl urea; NADP –nicotinamide adenine dinucleotide phosphate; Pi–orthophosphate‐ion; PMS–N‐methyl‐phenazonium sulphate or phenazinemethosulphate;PS–1–photosystem 1;PS–2–photosystem 2.

Abstract— Action spectra of phenazinemethosulfate (PMS)‐catalyzed photophosphorylation, measured under aerobic conditions without added reductants, show the involvement of both photosystem 2 (PS II) and photosystem 1 (PS I). Addition of low concentrations 3‐(3,4)dichlorophenly‐l, 1‐dimethylurea (DCMU) changes the shape of the curves and causes formation of typical PS II action spectra. This can be understood by the partial inhibition of PS II electron flow by DCMU, which therefore becomes rate‐limiting. The presence of DCMU causes a strong decrease of photochemical activity in these spectra at 638, 660 and 690 nm, which we attribute to decreasing activity of PS I pigments. Curve analysis of the action spectra suggests the presence of different Chi forms at room temperature peaking at: 641.6; 650.3; 661.2; 669.8; 677.1; 684.0 and 690.6 nm. Comparison of our data with published data from low temperature absorption spectra indicates that lowering the temperature causes a decrease in halfwidth of the absorption bands but no significant change in peak position. Our data support the hypothesis of French and co‐workers that there are four universal forms of Chi a also at 25â°C. Careful investigation of data in literature does not give evidence that the pigment peaking at 641 nm is a second form of Chi b, as is sometimes assumed. Because our action spectra show two small but distinct maxima at703–705 and718–720 nm when PS II is rate‐limiting, we also measured action spectra of PS I (reduced PMS + DCMU) and PS II (ferricyanide‐ions + 2,5‐dibromo‐3‐methyl‐6‐isopropylbenzoquinone) separately. They show, in agreement with published data, that PS I is operating more efficiently in the spectral region690–725 nm than PS II. The Chi form peaking at 690 nm is absent in the PS II action spectrum, whereas this curve has two small but distinct maxima around703–705 and718–720 nm, respectively, which are also present in the PS I action spectrum. We, therefore, suggest that light absorbed by these pigments can be transferred to either of the two photosystems, and not to PS I exclusively as is sometimes assumed. ATP,adenosine–5′‐triphosphate; BSA, bovine serum albumin; Chi, chlorophyll; DBMIB, 2,5‐dibromo‐3‐methyl‐6‐isopropylbenzoquinone; DCMU, 3‐(3,4)‐dichlorophenyl‐1,1‐dimethyl urea; NADP, nicotinamide adenine dinucleotide phosphate; Pi orthophosphate‐ion; PMS, JV‐methylphenazonium sulphate or phenazinemethosulphate; PS I, photosystem 1; PS II, photosystem 2.