Interaction between light and chilling temperature on the inhibition of photosynthesis in chilling-sensitive plants*

Abstract. Fully expanded leaves of 25°C grown Phaseolus vulgaris and six other species were exposed for 3 h to chilling temperatures at photon flux densities equivalent to full sunlight. In four of the species this treatment resulted in substantial inhibition of the subsequent quantum yield of CO₂ uptake, indicating reduction of the photochemical efficiency of photosynthesis. The extent of inhibition was dependent on the photon flux density during chilling and no inhibition occurred when chilling occurred at a low photon flux density. No inhibition occurred at temperatures above 11.5°C, even in the presence of the equivalent of full sunlight. This interaction between chilling and light to cause inhibition of photosynthesis was promoted by the presence of oxygen at normal air partial pressures and was unaffected by the CO₂ partial pressure present when chilling occurred in air. When chilling occurred at low O₂ partial pressures, CO₂ was effective in reducing the degree of inhibition. Apparently, when leaves of chilling-sensitive plants are exposed to chilling temperatures in air of normal composition then light is instrumental in inducing rapid damage to the photochemical efficiency of photosynthesis.     

Response of tomato plants to chilling stress in association with nutrient or phosphorus starvation

The experiments were conducted on two tomato cultivars: Garbo and Robin. Mineral starvation due to plant growth in 20-fold diluted nutrient solution (DNS) combined with chilling reduced the rate of photosynthesis (P _N) and stomatal conductance (g) to a greater extent than in plants grown in full nutrient solution (FNS). In phosphate-starved tomato plants the P _N rate and stomatal conductance decreased more after chilling than in plants grown on FNS. In low-P plants even 2 days after chilling the recovery of CO₂ assimilation rate and stomatal conductance was low. A resupply of phosphorus to low-P plants (low P + P) did not improve the rate of photosynthesis in non-chilled plants (NCh) but prevented P_N inhibition in chilled (Ch) plants. The greatest effect of P resupply was expressed as a better recovery of photosynthesis and stomatal conductance, especially in non-chilled low P + P plants. The F _v/F _m (ratio of variable to maximal chlorophyll fluorescence) decreased more during P starvation than as an effect of chilling. Supplying phosphorus to low-P plants caused the slight increase in the F _v/F _mratio. In conclusion, after a short-term chilling in darkness a much more drastic inhibition of photosynthesis was observed in nutrient-starved or P-insufficient tomato plants than in plants from FNS. This inhibition was caused by the decrease in both photochemical efficiency of photosystems and the reduction of stomatal conductance. The presented results support the hypothesis that tomato plants with limited supply of mineral nutrients or phosphorus are more susceptible to chilling.     

Determining the limitations and regulation of photosynthetic energy transduction in leaves

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.     

不同光强下细胞外三磷酸腺苷对菜豆叶片光化学反应特性的影响

三磷酸腺苷(ATP)不但分布在细胞内部, 而且广泛存在于动物和植物细胞的细胞外基质中。细胞外ATP (eATP)可与细胞膜表面相应的受体结合并激发细胞内的第二信使, 从而调节细胞的多种生理学功能。但目前对于eATP是否也能对植物的光合作用产生影响则研究较少。该文以菜豆(Phaseolus vulgaris)叶片为实验材料, 研究了在不同光强下eATP对菜豆叶片叶绿素荧光特性和光合放氧速率的影响。结果显示, 随着光强的增加, 叶片的光适应下最大光化学效率(F_v′/F_m′)、光系统II (PSII)实际光化学效率(Y(II))、光化学猝灭系数(q_P)均呈现下降趋势, 而电子传递速率(ETR)、非光化学猝灭系数(q_N)以及调节性能量耗散的量子产量(Y(NPQ))随着光强的增加呈上升趋势。与对照相比, eATP的处理可以显著提高菜豆叶片PSII的潜在最大光化学效率(F_v/F_m)、Y(II)、q_P、ETR和光合放氧速率; 但eATP的处理对F_v′/F_m′、q_N以及Y(NPQ)没有显著影响。AMP-PCP (β,γ-亚甲基三磷酸腺苷, eATP细胞外受体的抑制剂)的处理显著降低了F_v/F_m、F_v′/F_m′、Y(II)、ETR和光合放氧速率, 同时也显著增加了q_N以及Y(NPQ)的水平。以上结果显示, 植物eATP水平的变化对植物光合作用的光化学反应有着重要的影响。     

Characterization of a 31 kDa polypeptide that accumulates in the light-harvesting apparatus of maize leaves during chilling

A 31 kDa polypeptide that accumulates in the thylakoids when maize leaves are chilled to 5°C in the light is characterized using monoclonal antibodies and analyses of chlorophyll-protein complexes. This polypeptide reacted with a monoclonal antibody, MLH2, that was specific for the 28 kDa polypeptide of the light-harvesting complex (LHCII) of pea leaves. On chilling leaves the appearance of a chlorophyll-protein complex having an apparent molecular weight of 31 kDa coincided with the appearance of a 31 kDa polypeptide and a decrease in the 29 kDa chlorophyll-protein, CP29. Returning the leaves to 25°C for 1 h produced a loss of both the 31 kDa chlorophyll-protein and 31 kDa polypeptide from the thylakoids, and an increase in the amount of CP29. Breakdown of the 31 kDa polypeptide in vitro was Mg²⁺-dependent and inhibited by EDTA and transition metal ions. It is suggested that the 31 kDa polypeptide may be a precursor of the apoprotein of CP29 and can bind chlorophyll. The appearance of the 31 kDa polypeptide correlated with a marked change in the 77 K fluorescence emission spectra of isolated LHCII particles, which did not revert with the disappearance of the 31 kDa on returning the leaves to 25°C for 1 h. The physiological significance of this spectral perturbation is discussed.     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Will rising CO2 protect plants from the midday sun? A study of photoinhibition of Quercus myrtifolia in a scrub‐oak community in two seasons

Over a large part of the photoperiod, light energy absorbed by upper canopy leaves saturates photosynthesis and exceeds the energetic requirements for light‐saturated linear electron flow through photosystem II (J_PSII), so that photoinhibition results. From a theoretical consideration of the response of light‐saturated photosynthesis to elevated atmospheric CO₂ partial pressure (pCO₂) it may be predicted that, where light‐saturated photosynthesis is Rubisco‐limited, an increase in pCO₂ will stimulate J_PSII. Therefore, the proportion of absorbed quanta dissipated photochemically will increase and the potential for photoinhibition of photosynthesis will decrease. This was tested by measuring modulated chlorophyll a fluorescence from Quercus myrtifolia Willd. growing in the field in open‐top chambers, at either current ambient or elevated (ambient + 35 Pa) pCO₂ on Merritt Island, Florida, USA. During spring and summer, light‐saturated photosynthesis at current ambient pCO₂ was Rubisco‐limited. Consistent with theoretical prediction, J_PSII was increased and photoinhibition decreased by elevated pCO₂ in spring. In the summer, when growth had largely ceased, an acclimatory decrease in the maximum Ribulose 1,5 bisphosphate saturated carboxylation capacity (V_{c max}) removed the stimulation of J_PSII seen in the spring, and photoinhibition was increased in elevated pCO₂. It is concluded that, for Q. myrtifolia growing in the field, the effects of elevated pCO₂ on J_PSII and photoinhibition will reflect seasonal differences in photosynthetic acclimation to elevated pCO₂ in a predictable manner.     

Experimental evidence that evolutionary relatedness does not affect the ecological mechanisms of coexistence in freshwater green algae

The coexistence of competing species depends on the balance between their fitness differences, which determine their competitive inequalities, and their niche differences, which stabilise their competitive interactions. Darwin proposed that evolution causes species' niches to diverge, but the influence of evolution on relative fitness differences, and the importance of both niche and fitness differences in determining coexistence have not yet been studied together. We tested whether the phylogenetic distances between species of green freshwater algae determined their abilities to coexist in a microcosm experiment. We found that niche differences were more important in explaining coexistence than relative fitness differences, and that phylogenetic distance had no effect on either coexistence or on the sizes of niche and fitness differences. These results were corroborated by an analysis of the frequency of the co‐occurrence of 325 pairwise combinations of algal taxa in > 1100 lakes across North America. Phylogenetic distance may not explain the coexistence of freshwater green algae.     

In search of a reversible stage of photoinhibition in a higher plant: No changes in the amount of functional Photosystem II accompany relaxation of variable fluorescence after exposure of lincomycin-treated Cucurbita pepo leaves to high light

Pumpkin (Cucurbita pepo L.) leaves in which chloroplast protein synthesis was inhibited with lincomycin were exposed to strong photoinhibitory light, and changes in F_O, F_M, F_V/F_M and in the amount of functional Photosystem II (O₂ evolution induced by saturating single-turnover flashes) were monitored during the high-light exposure and subsequent dark or low-light incubation. In the course of the photoinhibitory illumination, F_M, F_V/F_M and the amount of functional PS II declined continuously whereas F_O dropped rapidly to some extent and then slowly increased. If the experiments were done at room temperature, termination of the photoinhibitory illumination resulted in partial relaxation of the F_V/F_M ratio and in an increase in F_O and F_M. The relaxation was completed in 10–15 min after short-term (15 min) photoinhibitory treatment but continued 30–40 min if the exposure to high light was longer than 1 h. No changes in the amount of functional PS II accompanied the relaxation of F_V/F_M in darkness or in low light, in the presence of lincomycin. Transferring the leaves to low temperature (+4°C) after the room-temperature illumination (2 h) completely inhibited the relaxation of F_V/F_M. Low temperature did not suppress the relaxation if the photoinhibitory illumination had also been done at low temperature. The results indicate that illumination of lincomycin-poisoned pumpkin leaves at room temperature does not lead to accumulation of a reversibly photoinactivated intermediate.Abbreviations F_O, F_M chlorophyll fluorescence with all reaction centres open or closed, respectively - F_V variable fluorescence (F_V=F_M–F_O) - LHC Light-harvesting complex - PS II Photosystem II - Q_A, Q_B primary and secondary quinone electron acceptors of PS II, respectively - qN_E, qN_T, qN_I non-photochemical quenching due to high-energy state, state transition or photoinhibition, respectively     

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1)

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF₁) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF₁ is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg²⁺ -dependent ATPase activity of the isolated CF₁.     

Photosynthate partitioning and nodule formation in soybean plants that received red or far-red light at the end of the photosynthetic period

The influence of row orientation on spectral distribution of light received by growing soybean [ Glycine max (L.) Merr. (cv. Coker 338)] plants was measured under field conditions, and light spectrum effects on photosynthate partitioning were studied under controlled environments. Light received by leaves under field conditions differed among those grown in north-south vs east-west oriented rows. In morning and late afternoon, the far-red/ red ratio received by leaves at the surface of the canopy differed about 3-fold from the east to west sides of north-south rows, but only 1.3-fold from the south to north sides of east-west rows.
In controlled environments, brief exposures to red or far-red light at the end of the photosynthetic period influenced partitioning of photosynthate among leaves, stems and roots. The top/root ratios differed significantly between the red and far-red treated plants. Red treated plants partitioned less photosynthate to stems and more to roots than did those treated with far-red. Also, plants with larger root systems developed more nodules. Phytochrome effects on photosynthate partitioning between tops and roots may influence yield of soybean plants grown in soils with low water-holding capacity.     

Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation

Thiol modulation of the chloroplast protonmotive ATPase (CF₀-CF₁) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall $\text{P}\text{2}\text{e}$ ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in $\text{P}\text{2}\text{e}$ ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH₄Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in $\text{P}\text{2}\text{e}$ ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and $\text{P}\text{2}\text{e}$ ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF₀-CF₁ increased the observed Michaelis constant for ADP (K_m(ADP)) and the apparent maximum rate (V_app of photophosphorylation by the same factor, so that ratio $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was not altered. $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF₀-CF₁ is presented to explain the dependence of α on ΔpH. Thiol modulation of CF₀-CF₁ is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.     

In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux

Xu et al. [Biochem. Biophys. Res. Commun. 305 (2003) 941] reported that, when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 μM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually. The authors concluded that the intracellular ethidium bromide “induced” either its degradation or its efflux through the assembly of unknown efflux pumps. We found, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under these conditions, indicating the absence of its degradation. Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.     

Equilibration of methionine into the Met-tRNA pool of rabbit reticulocyte lysates: Additional evidence that deacylation of ribosomal subunit-bound Met-tRNAf does occur in heme-deficiency

We have measured the rate of equilibration of [³⁵S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301–305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNA_f that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

A gene trap mutation of a murine homolog of the Drosophila stem cell factor Pumilio results in smaller testes but does not affect litter size or fertility

Members of the Pumilio (also called PUF) gene family belong to a class of highly conserved developmental regulators that are present in both flies and humans. Much is known about the function of Pumilio genes in invertebrate development, in particular their role as stem cell factors required for maintenance and/or self-renewal of germline stem cells in Drosophila and Caenorhabditis elegans. It remains unknown whether Pumilio genes are also required for development in mammals; however, several lines of evidence suggest similar functions based on extensive sequence homology, similar RNA-binding properties to their invertebrate counterparts and well-documented interactions with germ cell factors required for fertility. Here we report characterization of a gene trap mutation that disrupts the mouse Pumilio-2 (Pum2) gene. Our data confirm that Pumilio-2 is expressed most abundantly in germ cells with the highest expression in undifferentiated gonocytes and spermatogonia. Furthermore, the mutation in Pum2 results in significantly smaller testes although the mutants are otherwise viable and fertile. In addition, we observed no stronger reproductive defects on a genetic background homozygous for a Pum2 null mutation and heterozygous for a Dazl mutation than Pum2 mutant alone. Thus, as in C. elegans where single members of the Pumilio gene family are dispensable for reproductive development and viability, this individual member of the Pumilio gene family in mice is also not essential for reproduction or viability.     

In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly

The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic conditions, is not suitable for high‐throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 3000 hygromycin‐resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.