Jeff A. Nemson

Damage to functional components and partial degradation of Photosystem II reaction center proteins upon chloroplast exposure to ultraviolet-B radiation

Abstract Exposure of thylakoid membranes to ultraviolet-B radiation caused inhibition of semiquinone anion formation at Q A , inhibition of plastoquinone photoreduction and lower rates of Photosystem II electron-transport to artificial electron acceptors. The amplitude of pheophytin photoreduction was unaffected by the UV-B treatment, suggesting lack of a UV-B adverse effect on the primary charge separation reaction between the photochemical reaction center P680 and pheophytin. Under the experimental conditions employed, approx. 50% inhibition in Q A photoreduction and in the variable to maximal fluorescence ratio ( F / F max ) was observed. However, plastoquinone photoreduction was lowered by about 65% and electron-transport measurements from H 2 O to dichlorophenol indophenol were inhibited by 70–90% in the UV-B treated thylakoids. Rates of electron-transport through PS II could not be restored upon inclusion of artificial donors such as diphenyl carbazide or hydroxylamine. The results suggest a UV-B-induced damage to the primary quinone acceptor Q A and impairment in the function of plastoquinone in the thylakoid membrane. SDS-PAGE and immunoblot analysis of UV-B-exposed thylakoids revealed the appearance of small quantities of polypeptide fragments (13,11 and 5 kDa) from the Photosystem II reaction-center proteins. We suggest multiple independent targets of UV-B-irradiance in Photosystem II and point to plastoquinone, in its many different configurations in the thylakoid membrane, as a primary UV-B photosensitizer molecule.

Chromatic regulation inChlamydomonas reinhardtii alters photosystem stoichiometry and improves the quantum efficiency of photosynthesis

The work addressed the adjustment of the photosystem ratio in the green algaChlamydomonas reinhardtii. It is shown that green algae, much like cyanophytes and higher plants, adjust and optimize the ratio of the two photosystems in chloroplasts in response to the quality of irradiance during growth. Such adjustments are compensation reactions and helpC. reinhardtii to retain a quantum efficiency of oxygen evolution near the theoretical maximum. Results show variable amounts of PS I and a fairly constant amount of PS II in chloroplasts and suggest that photosystem stoichiometry adjustments, occurring in response to the quality of irradiance during plant growth, are mainly an adjustment in the concentration of PS I. The work delineates chromatic effects on chlorophyll accumulation in the chloroplast ofC. reinhardtii from those pertaining to the regulation of the PS I/PS II ratio. The detection of the operation of a molecular feedback mechanism for the PS I/PS II ratio adjustment in green algae strengthens the notion of the highly conserved nature of this mechanism among probably all oxygen evolving photosynthetic organisms. Findings in this work are expected to serve as the basis of future biochemical and mutagenesis experiments for the elucidation of the photosystem ratio adjustment in oxygenic photosynthesis.

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.

Photosystem stoichiometry and chlorophyll antenna size in Dunaliella salina (green algae)

Abstract The photochemical apparatus organization in the green alga Dunaliella salina was investigated. Photosystem (PS) stoichiometry was estimated from direct quantitations of the reaction center P-700 (PS I) and of the primary electron acceptors pheophytin and Q A (PS II). A PS II/PS I ratio of 1.4/1.0 was deduced. Analysis of PS II activity revealed heterogeneity both in the chlorophyll (Chl) antenna size of the PS II unit and in the electron-transport process from Q A to plastoquinone in the thylakoid membrane. Antenna size measurements indicated the presence of PS II α (55% of all PS II) with about 570 Chl ( a + b ) molecules, and of PS II β (45% of all PS II) with about 150 Chl ( a + b ) molecules per reaction center. By comparison, PS I contained about 220 Chl ( a + b ) molecules per reaction center. Analysis of the electron-transport reactions on the reducing side of PS II revealed that approximately 25% of all PS II centers, although photochemically competent, were unable to transfer electrons from the primary quinone acceptor Q A to the secondary quinone Q B (PS II-Q B -nonreducing). Based on the fluorescence yield and exponential induction kinetics displayed by PS II-Q B -nonreducing, we suggest that the latter is a subpopulation of PS II β in Dunaliella salina . It is speculated that PS II-Q B -nonreducing centers represent newly synthesized and/or repaired PS II centers which have not yet established a functional interaction between Q A and Q B .

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, QA, to the secondary-quinone electron acceptor, QB (QB-nonreducing centers). Both complements of the PS II Chl a−b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a β-type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS IIβ units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma QB-nonreducing form in the dark, to a QB-reducing form in the light. Concomitant with this change, PS IIβ units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS IIα form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the QA-QB electron-transfer reaction which is followed by association of the PS IIβ unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS IIα) in the membrane of the grana partition region.

Microwave Protocols for Paraffin Microtechnique and In Situ Localization in Plants

: We have developed a microwave protocol for a paraffin-embedding microtechnique of the shoot apical meristem of Zea mays and have successfully applied this protocol to other plant tissues. This protocol decreases the time required for all aspects of microtechnique tissue processing, including fixation (24 hr to 15 min), dehydration (73 hr to 10 min), and infiltration (96 hr to 3 hr). Additionally, the time required to adhere paraffin ribbons to gelatin-coated slides and for the Johansons safranin O, fast green FCF staining protocol has been significantly decreased. Using this technique, the quality of tissue preservation and subsequent in situ localization of knotted mRNA was increased by using microwaves.

GRAVITAXIS IN CHLAMYDOMONAS REINHARDTII: CHARACTERIZATION USING VIDEO MICROSCOPY AND COMPUTER ANALYSIS

We characterized the gravitactic behavior of Chlamydomonas reinhardtii, a unicellular green alga, using a computer‐analysis system in order to study directional swimming. The effects of the calcium‐channel inhibitors gadolinium and diltiazem on graviorientation and swimming speed were examined. In addition, we studied directional swimming in the ptx1 strain of C. reinhardtii, a flagellar dominance mutant. Results indicate that Chlamydomonas reorients for gravitactic swimming through a mechanism different from the calcium‐mediated pathway believed to be involved in gravity transduction in higher plants. We suggest that calcium‐mediated gravitaxis originated in an organism that was more evolutionarily advanced than Chlamydomonas.

Characterization of a 160 kD Photosystem II reaction center complex isolated from photoinhibited Dunaliella salina thylakoids

Photoinhibition in the green alga Dunaliella salina is accompanied by the formation of inactive Photosystem II reaction centers. In SDS-PAGE analysis, the latter appear as 160 kD complexes. These complexes are structurally stable, enough to withstand re-electrophoresis of excised gel slices from the 160 kD region. Western blot analyses with specific polyclonal antibodies raised against the D1 or D2 reaction center proteins provided evidence for the presence of both of these polypeptides in the re-electrophoresed 160 kD complex. Incubation of excised gel slices from the 160 kD region, under aerobic conditions at 4°C for a prolonged period of time, caused a break-up of the 160 kD complex into a ∽52 kD D1-containing and ∽80 and ∽26 kD D2-containing pieces. Western blot analysis with polyclonal antibodies raised against the apoproteins of CPI (reaction center proteins of PS I) did not show cross-reaction either with the 160 kD complex or with the ∽52, ∽80 and ∽26 kD pieces. The results show the presence of both D1 and D2 in the 160 kD complex and strengthen the notion of a higher molecular weight D1- and D2-containing complex that forms upon disassembly of photodamaged PS II units.

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.

Light-Induced Oxidation-Reduction Reactions of Photosystem II in Dichlorophenyl-dimethyl Urea (DCM U) Inhibited Thylakoids

Abstract Illumination of thylakoid membranes in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea (DCMU) causes the reduction of the primary quinone acceptor QA of photosystem II (PS II) and the storage of a positive charge on the donor side of the photochemical reaction center. These oxidation-reduction reactions are accompanied by characteristic changes of absorbance in the ultra-violet region of the spectrum. The PS II-related absorbance difference spectra (250 -350 nm) were compared in control and hydroxylamine-treated thylakoid membranes, and in thylakoids suspended in the presence of carbonyl cyanide-p-(trifluoromethoxy)- phenylhydrazone (FCCP). The light minus dark difference spectra were dominated by the Q-A minus QA difference spectrum. Qualitatively, the three spectra were identical in the 300 - 350 nm region, however, they showed distinct differences in the 250 - 300 nm region. The latter arose because of different contributions from the donor side of PS II in the thylakoid membrane of the three samples. The result suggested that FCCP acts as the ultimate electron donor in DCMU - poisoned chloroplasts. Therefore, the absorbance difference spectrum in the presence of FCCP reflected a contribution from the Q-A minus QA component only. Deconvolution of the absorbance difference spectra of control and hydroxylamine-treated thylakoids yielded difference spectra attributed to the oxidation of a component on the donor side of PS II. This component did not conform with the known Mn(III) → Mn(IV) transition. Rather, it indicated the oxidation of a modified form of Mn in the presence of DCMU , probably a Mn(II) → Mn(III) transition. The results are discussed in terms of the use of DCMU - poisoned thylakoid membranes in the quantitation of the primary quinone acceptor QA by spectrophotometric approaches.