The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants.

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.

Decreased Hill reaction rates and slow turnover of transitory starch in the obligate shade plant Panax quinquefolius L. (American ginseng).

To identify physiological processes that might limit photosynthesis in Panax quinquefolius L. (American ginseng) a comparison has been made with Panax ginseng C.A. Meyer (Korean ginseng), Pisum sativum L. (pea) and Spinacia oleracea L. (spinach). The quantum yield of oxygen evolution in intact leaves and isolated thylakoid membranes was found to be smaller in ginseng than in pea or spinach. However, the number of photosystem II (PSII) centers on a chlorophyll basis was found to be similar in all species. This suggests that ginseng thylakoid membranes possess relatively more inactive PSII centers than thylakoids of pea and spinach when grown under similar conditions. Unexpectedly, whole-chain electron transport from water to methyl viologen, and partial photosystem I reactions, demonstrated that electron transport rates to methyl viologen were anomalously low in P. quinquefolius and P. ginseng. Additionally, at elevated light intensities, intact leaves of P. quinquefolius were more susceptible to lipid peroxidation than pea leaves. In plants grown at a light intensity of 80 micro mol photons m(-2) s(-1) the levels of fructose and starch were higher in both ginseng species than in pea or spinach. Significantly, the level of starch in P. quinquefolius was relatively constant throughout the entire 12 h/12 h light/dark cycle and remained high after an extended dark time of 48 h. In addition, P. quinquefolius had lower activities of alpha-amylase and beta-amylase than P. ginseng, pea and Arabidopsis thaliana (L.) Heynh. The significance of the elevated levels of leaf starch in P. quinquefolius remains to be determined. However, the susceptibility of P. quinquefolius to photoinhibition may arise as a consequence of a reduced fraction of active PSII centers. This may result in the normal dissipative mechanisms in these plants becoming saturated at elevated, but moderate, light intensities.

Diurnal variation of photosynthesis and photoinhibition in tea: effects of irradiance and nitrogen supply during growth in the field.

Diurnal changes in the rate of photosynthesis (A) of mature tea (Camellia sinensis (L.) O. Kuntze) bushes grown at high elevation in the field in Sri Lanka, were related to environmental conditions. Bushes were either unshaded, receiving 100% of incident photosynthetically active radiation (PAR), moderately shaded, (65% PAR) or heavily shaded (30% PAR). These treatments were combined with nitrogen fertilizer applications of 0, 360 and 720 kg ha(-1) year(-1). When recently fully expanded leaves were measured under the growing conditions on bright, clear days from dawn to dusk, A was greatest in the morning with increasing radiation between approximately 8 h and 10 h. Stomatal conductances (g(s)) and substomatal carbon dioxide concentrations (C(i)) were then large, leaf temperatures (T(L)) cool, and saturated water vapour deficits (VPD) small. However, as the irradiance, T(L) and VPD increased towards midday, A, g(s), photochemical quenching, and C(i) decreased, and non-photochemical quenching increased. In the late afternoon, irradiance, T(L) and VPD fell, but despite the relatively large increase in g(s) and C(i), A remained low; however, it recovered overnight. The zero-N treatment decreased total-N content of leaves by 50% and A by c. 20% (not significant). Leaves of unshaded plants receiving least N had significantly (P<0.05) smaller A and greater total sugar content than shaded but with abundant N, A and sugars did not differ between shade treatments. Analysis of the responses of A to environment in the morning compared to the afternoon, and of chlorophyll fluorescence, suggests that A was photoinhibited as a consequence of greatly increased PAR, whilst decreasing g(s) (related to changes in PAR, VPD and T(L)) caused C(i) to fall. End-product inhibition of A is not consistent with decreased C(i). Inhibition of A as a result of photoinhibition was minimized, but not eliminated, by abundant N. Interactions between factors regulating A in tea are discussed.

Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803.

Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp. PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T. W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I. R., Bryant, D. A., Jones, A. D., Golbeck, J. H., and Chitnis, P. R. (2000) J. Biol. Chem. 275, 8523-8530. We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio. PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ. The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action. The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ. Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site. Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact. Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3. Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group. These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells.

An evaluation of the potential triggers of photoinactivation of photosystem II in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model.

Photoinactivation of photosystem II (PS II) is a light-dependent process that frequently leads to break-down and replacement of the D1 polypeptide. Photoinhibition occurs when the rate of photoinactivation is greater than the rate at which D1 is replaced and results in a decrease in the maximum efficiency of PS II photochemistry. Downregulation, which increases non-radiative decay within PS II, also decreases the maximum efficiency of PS II photochemistry and plays an important role in protecting against photoinhibition by reducing the yield of photoinactivation. The yield of photoinactivation has been shown to be relatively insensitive to photosynthetically active photon flux density (PPFD). Formation of the P680 radical (P680+), through charge separation at PS II, generation of triplet-state P680 (3P680*), through intersystem crossing and charge recombination, and double reduction of the primary stable electron acceptor of PS II (the plastoquinone, Q(A)) are all potentially critical steps in the triggering of photoinactivation. In this paper, these processes are assessed using fluorescence data from attached leaves of higher plant species, in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model. It is shown that the yield of P680+ is very sensitive to PPFD and that downregulation has very little effect on its production. Consequently, it is unlikely to be the trigger for photoinactivation. The yields of 3P680* generated through charge recombination or intersystem crossing are both less sensitive to PPFD than the yield of P680+ and are both decreased by down regulation. The yield of doubly reduced Q(A) increases with incident photon flux density at low levels, but is relatively insensitive at moderate to high levels, and is greatly decreased by downregulation. Consequently, 3P680* and doubly reduced Q(A) are both viable as triggers of photoinactivation.

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band.

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.

The rates of proton uptake and electron transfer at the reducing side of photosystem II in thylakoids.

Proton and electron transfer at the reducing side of photosystem II of green plants was studied under flashing light, the former at improved time resolution by using Neutral red. The rates of electron transfer within QAFeQB were determined by pump-probe flashes through electrochromic transients. The extent of proton binding was about 1 H+/e-. The rates of proton transfer were proportional to the concentration of Neutral red (collisional transfer), whereas the rates of electron transfer out of QA- and from QAFeQB- to the cytochrome b6f complex were constant. The half-rise times of electron transfer (tau e) and the apparent times of proton binding (tau h) at 30 microM Neutral red were: QA- --> FeIIIQB (tau c < or = 100 microseconds, tau h = 230 microseconds); QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 620 microseconds, tau h = 310 microseconds).

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Why does the light-gradient photovoltage from photosynthetic organelles show a wavelength-dependent polarity?

The light-gradient photovoltage from photosynthetic organisms and organelles is thought to arise from the primary charge separation in the reaction centers. The current explanation of the effect is the stronger excitation of the membrane side of a vesicle facing the light source than the one on the opposite side. Together with the known orientation of reaction centers, this explanation predicts unequivocally the polarity of the photovoltage. However, a polarity opposite to the one expected has often been reported. A dependence of the polarity on the wavelength has been published but no explanation was given (Gräber, P., and H.-W. Trissl. 1981. FEBS Lett. 123:95-99). Here we report on a theoretical treatment of light propagation and interference in pigmented and nonpigmented multilayers. A model calculation is carried out for a pair of membranes, demonstrating the wavelength-dependent light distribution as well as the relative photovoltage and its polarity. When the membranes contain no chromophores or when the absorption coefficient is low, the predicted polarity to that expected from a simple macroscopic absorption behavior. The model is tested by comparing new photovoltage data obtained at 532 nm as well as in the blue and red absorption bands of chlorophyll in chloroplasts. It is concluded that outside the main absorption bands the amplitude and polarity of the photovoltage is determined by the ratio of the refractive indices of the membrane and the medium.

Photoinduced degradation of the D1 polypeptide in isolated reaction centers of photosystem II: evidence for an autoproteolytic process triggered by the oxidizing side of the photosystem.

When the isolated D1/D2/cytochrome b559 complex was exposed to bright light, a distinctive pattern of D1 polypeptide fragments was observed under both aerobic and anaerobic conditions. The major degradation product had an apparent molecular mass of 24 kDa, while other fragments were detected at 17, 14, and 10 kDa by immunoblotting. This pattern was observed when the electron acceptors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or silicomolybdate were present during illumination. It is known that these conditions stabilize P680+ chlorophyll and bring about the photooxidation and destruction of pigments in the reaction center, particularly chlorophyll absorbing at 670 nm and beta-carotene. When P680+ was not allowed to accumulate, either by omission of an electron acceptor or by addition of both an electron donor (Mn2+) and an acceptor, no breakdown fragments were observed. In the former case, however, some degradation of the D1 and D2 polypeptides did occur. Under conditions that gave rise to the characteristic D1 breakdown pattern, the D2 polypeptide was also degraded to specific fragments detected at about 29 and 21 kDa by immunoblotting. The results indicate that the photoinduced degradation of D1 (and D2) does not involve exogenous proteases but is most likely an autoproteolytic process. Moreover, our data indicate that the photochemical damage giving rise to D1 and D2 degradation occurs on the oxidizing rather than the reducing side of photosystem II and involves photooxidation of the accessory pigments. The results are discussed in terms of D1 and D2 turnover and photoinhibition.