The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II     

Organization of the photosynthetic units,and onset of electron transport and excitation energy distribution in greening leaves

The development and organization of the Photosynthetic units follow a step-wise assembly process. First the core complexes of the PSI and PSII units are formed, followed by their light-harvesting components; then an assembly process of these components into supramolecular structures takes place. Parallel to this, the control of excitation energy distribution between the two photosystems is established. This control is attributed to the modulation of the PSI unit effective cross section, which is possible only when LHC-I is formed and assembled into CPIa. Parallel to the formation of PSI and PSII, the electron carriers are synthesized and the electron transport chain is assembled. The number of PSII units operating per electron transport chain remains constant throughout development and equal to that of the mature chloroplast, but the number of PSI units per chain varies with PSII unit size. During development, when the rate of Chla synthesis is low, relative to the other thylakoid components, or is completely stopped, then the newly formed or preexisting LHC-I and LHC-II proteins are digested and their Chla is used for the formation of PS core complexes.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

Circadian expression of the light-harvesting protein of Photosystem II in etiolated bean leaves following a single red light pulse: Coordination with the capacity of the plant to form chlorophyll and the thylakoid-bound protease

The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665–672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.Abbreviations Chl chlorophyll - CL continuous light - D dark - FR far-red light - LA levulinic acid - LHC II light-harvesting complex serving Photosystem II - PChl(ide) protochlorophyllide - PCR protochlorophyllide oxidoreductase - R red light     

Specificity of the chlorophyll-to-protein binding in the chlorophyll-protein complexes of the thylakoid

We tried to establish whether the chlorophyll-protein complexes of the thylakoid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, originate from real entities existing in vivo, or are mere artifacts of the sodium dodecyl sulfate solubilization procedure. Making use of the finding that etiolated leaves exposed to periodic light form selectively the chlorophyll-protein complexes CPI and CPa, while after transfer to continuous light they form in addition the light-harvesting complexes (J. H. Argyroudi-Akoyunoglou, Z. Feleki, and G. Akoyunoglou, 1971, Biochem. Biophys. Res. Commun.45, 606–614; J. H. Argyroudi-Akoyunoglou and G. Akoyunoglou 1979, FEBS Lett.104, 78–84) we tried to see whether the latter complexes contain newly formed chlorophyll. We labeled the chlorophyll a formed in periodic light with δ-[¹⁴C]aminolevulinic acid, and determined the specific radioactivity of chlorophyll in the complexes formed before or after transfer to continuous light. We found that the light-harvesting complexes contain primarily newly formed and nonradioactive chlorophyll. The results suggest that (i) the chlorophyll a of CPI and CPa formed in periodic light does not exchange with that of the light-harvesting complexes formed after transfer to continuous light. (ii) The light-harvesting complexes formed after transfer to continuous light contain primarily newly formed chlorophylls a and b. (iii) The binding of chlorophyll to protein in the complexes is specific and not an artifact of the sodium dodecyl sulfate action. (iv) As the thylakoid membrane grows and differentiates, the chlorophyll synthesized binds on the apoproteins of the complexes in a stepwise manner.     

Correlation between cation-induced formation of heavy subchloroplast fractions and cation-induced increase in chlorophyll a fluorescence yield in Tricine-washed chloroplasts

A good correlation exists between the extent of thylakoid aggregation (grana reconstitution) and the increase in the chlorophyll a fluorescence yield (F_DCMU; DCMU = 3-(3′,4′-dichlorophenyl)-1, 1-dimethyl urea) caused by the addition of monovalent or divalent cations to low-salt disorganized (agranal) chloroplasts. The extent of grana stacking was monitored by the yield of heavy subchloroplast fractions after digitonin disruption of chloroplasts. A good correlation of the cation effect on both parameters was also found in light subchloroplast fractions (10,000g supernatants) obtained from sonicated “low-salt” Tricine-suspended pea chloroplasts. Addition of cations to the agranal protochloroplasts of etiolated pea or bean leaves exposed to periodic light-dark cycles, suspended in low-salt Tricine buffer, does not affect formation of heavy subchloroplast fractions, nor does it affect their chlorophyll a fluorescence yield level (F_DCMU). The cation effect on the increase of the chlorophyll a fluorescence yield level seems to be due to the cation-induced thylakoid structural changes leading to grana stacking.     

Growth of grana from "primary" thylakoids in Phaseolus vulgaris

The plastids of young dark-grown bean leaves, exposed to periodiclight are agranal, devoid of chlorophyll b and contain primarythylakoids and chlorophyll a. Transfer of these plants to continuousillumination results in synthesis of new chlorophyll a, chlorophyllb and grana. This study was done in order to study whether andhow the grana are formed from preexisting primary thylakoids.¹⁴C--aminolevulinic acid was used to label the chlorophyll aof the primary thylakoids, and its fate was studied after transferof the plants to continuous light. It was found that chlorophyll b and grana become ¹⁴C-labelled.The total radioactivity of chlorophyll b per bean increasedwith the parallel decrease of that of chlorophyll a. All subchloroplastfractions, obtained after digitonin disruption of chloroplasts,contained chlorophyll a of equal specific radioactivity. Thespecific radioactivity of chlorophyll b was lower than thatof chlorophyll a, and, in addition, it was lower in the granathan in the stroma lamellae fraction. The data suggest that chlorophyll b is formed from chlorophylla; the grana are formed by stacking of preexisting primary thylakoids;chlorophyll b is synthesized faster in the grana than the stromalamellae; the newly formed chlorophyll a molecules are distributedat random throughout the developing photosynthetic membraneand not on specific growing sites. (Received April 24, 1976; )