Entropy and biological systems: experimentally-investigated entropy-driven stacking of plant photosynthetic membranes

According to the Second Law of thermodynamics, an overall increase of entropy contributes to the driving force for any physicochemical process, but entropy has seldom been investigated in biological systems. Here, for the first time, we apply Isothermal Titration Calorimetry (ITC) to investigate the Mg21-induced spontaneous stacking of photosynthetic membranes isolated from spinach leaves. After subtracting a large endothermic interaction of MgCl2 with membranes, unrelated to stacking, we demonstrate that the enthalpy change (heat change at constant pressure) is zero or marginally positive or negative. This first direct experimental evidence strongly suggests that an entropy increase significantly drives membrane stacking in this ordered biological structure. Possible mechanisms for the entropy increase include: (i) the attraction between discrete oppositely-charged areas, releasing counterions; (ii) the release of loosely-bound water molecules from the inter-membrane gap; (iii) the increased orientational freedom of previously-aligned water dipoles; and (iv) the lateral rearrangement of membrane components.This work was supported consecutively by Australian Research Council grants (DP0664719 and DP 1093927)

The time course of photoinactivation of photosystem II in leaves revisited

Since photosystem II (PS II) performs the demanding function of water oxidation using light energy, it is susceptible to photoinactivation during photosynthesis. The time course of photoinactivation of PS II yields useful information about the process. Depending on how PS II function is assayed, however, the time course seems to differ. Here, we revisit this problem by using two additional assays: (1) the quantum yield of oxygen evolution in limiting, continuous light and (2) the flash-induced cumulative delivery of PS II electrons to the oxidized primary donor (P700+) in PS I measured as a ‘P700 kinetics area’. The P700 kinetics area is based on the fact that the two photosystems function in series: when P700 is completely photo-oxidized by a flash added to continuous far-red light, electrons delivered from PS II to PS I by the flash tend to re-reduce P700+ transiently to an extent depending on the PS II functionality, while the far-red light photo-oxidizes P700 back to the steady-state concentration. The quantum yield of oxygen evolution in limiting, continuous light indeed decreased in a way that deviated from a single-negative exponential. However, measurement of the quantum yield of oxygen in limiting light may be complicated by changes in mitochondrial respiration between darkness and limiting light. Similarly, an assay based on chlorophyll fluorescence may be complicated by the varying depth in leaf tissue from which the signal is detected after progressive photoinactivation of PS II. On the other hand, the P700 kinetics area appears to be a reasonable assay, which is a measure of functional PS II in the whole leaf tissue and independent of changes in mitochondrial respiration. The P700 kinetics area decreased in a single-negative exponential fashion during progressive photoinactivation of PS II in a number of plant species, at least at functional PS II contents ≥6 % of the initial value, in agreement with the conclusion of Sarvikas et al. (Photosynth Res 103:7–17, 2010). That is, the single-negative-exponential time course does not provide evidence for photoprotection of functional PS II complexes by photoinactivated, connected neighbours.The support of this study by an Australian Research Council Grant (DP1093827) awarded to W.S.C., a China Scholarship Council Fellowship to J.K., JSPS Postdoctoral Fellowships for Research Abroad (21-674) to R.O. and a Knowledge Innovation Programme of the Chinese Academy of Sciences Grant (KSCX2-EW-J-1) to D.-Y.F. is gratefully acknowledged

Intra-leaf gradients of photoinhibition induced by different color lights: Implications for the dual mechanisms of photoinhibition and for the application of conventional chlorophyll fluorometers

We studied how different color lights cause gradients of photoinhibition within a leaf, to attempt to resolve the controversy whether photon absorption by chlorophyll or Mn is the primary cause of photoinhibition, suggested by the excess-energy hypothesis or the two-step hypothesis, respectively. Lincomycin-treated leaf-discs were photoinhibited by white, blue, green or red light. Combining a micro-fiber fluorometer, a fiber-thinning technique and a micro-manipulator enabled us to measure the chlorophyll fluorescence signals within a leaf. Photoinhibition gradients were also compared with results from various conventional fluorometers to estimate their depth of signal detection. The severity of photoinhibition was in the descending order of blue, red and green light near the adaxial surface, and in the descending order of blue, green and red light in deeper tissue, which is correlated with the chlorophyll and Mn absorption spectrum, respectively. These results cannot be explained by either hypothesis alone. These data strongly suggest that (1) both the excess-energy and the two-step mechanisms occur in photoinhibition, and (2) fluorometers with red or blue measuring light give overestimated or underestimated Fv/Fm values of photoinhibited leaves compared with the whole tissue average, respectively; that is, they measured deeper or shallower leaf tissue, respectively

Impacts of LED spectral quality on leafy vegetables: Productivity closely linked to photosynthetic performance or associated with leaf traits?

The success of growing vegetables indoors requires the most appropriate selection of lighting spectrum. This mini review discusses the impacts of LED spectral quality on different leafy vegetables with a focus on the studies of Chinese broccoli (Brassica alboglabra), ice plants (Mesembryanthem crystallinum) and lettuce (Lactuca sativa L. cv. Canasta). For each species, plants exposed to different spectral LED lights were all under the same light intensity and same photoperiod. Chinese broccoli grown under red(R):blue(B)-LED ratio of 84:16 (16B) had the highest light-saturated photosynthetic CO2 assimilation rate (Asat) and stomatal conductance (gs sat) compared to plants grown under other R:B-LED ratios. It was also shown that 16B is the most appropriate selection for Chinese broccoli to achieve the highest shoot productivity with a rapid leaf number and leaf area development. The highest concentrations of photosynthetic pigments, soluble and Rubisco protein on a leaf area basis were also observed in 16B plants. The results conclusively affirmed that the highest productivity of Chinese broccoli grown under 16B is closely linked to the highest photosynthetic performance on a leaf area basis. For ice plants grown under R:B-LED ratios of 90:10 (10B), they had the highest shoot biomass with a faster leaf development compared to plants grown under other RB-LED combinations. However, there were no differences in Asat, gs sat, photosynthetic pigments, soluble and Rubisco proteins on a leaf area basis. In the case of lettuce plants, it was a surprise to observe that plants grown under 0B and 20G (20% green (G)-LED and 80% R-LED) had the highest shoot biomass, and largest total leaf area and light interception area but the lowest net maximal photosynthetic rate on a leaf area basis, compared to other plants. The combined RB-LED enhanced other photosynthetic parameters while 0B and 20G conditions had inhibitory effects on maximum quantum efficiency of PS II with lower photosynthetic pigments, total soluble protein and Rubisco protein. These results suggest that impacts of LED light quality on productivity of lettuce (L. sativa L. cv. Canasta) are closely linked to leaf traits not associated with photosynthetic performance on a leaf area basis.We thank the Singapore Millennium Foundation (SMF-Farming System) and Nanyang Technological University (NTU AcRF Tier 1, RP 1/18 HJ) for their financial support

Whole-tissue determination of the rate coefficients of photoinactivation and repair of photosystem II in cotton leaf discs based on flash-induced P700 redox kinetics

Using radioactively labelled amino acids to investigate repair of photoinactivated photosystem II (PS II) gives only a relative rate of repair, while using chlorophyll fluorescence parameters yields a repair rate coefficient for an undefined, variable location within the leaf tissue. Here, we report on a whole-tissue determination of the rate coefficient of photoinactivation k i , and that of repair k r in cotton leaf discs. The method assays functional PS II via a P700 kinetics area associated with PS I, as induced by a single-turnover, saturating flash superimposed on continuous background far-red light. The P700 kinetics area, directly proportional to the oxygen yield per single-turnover, saturating flash, was used to obtain both k i and k r . The value of k i , directly proportional to irradiance, was slightly higher when CO2 diffusion into the abaxial surface (richer in stomata) was blocked by contact with water. The value of k r , sizable in darkness, changed in the light depending on which surface was blocked by contact with water. When the abaxial surface was blocked, k r first peaked at moderate irradiance and then decreased at high irradiance. When the adaxial surface was blocked, k r first increased at low irradiance, then plateaued, before increasing markedly at high irradiance. At the highest irradiance, k r differed by an order of magnitude between the two orientations, attributable to different extents of oxidative stress affecting repair (Nishiyama et al., EMBO J 20: 5587–5594, 2001). The method is a whole-tissue, convenient determination of the rate coefficient of photoinactivation k i and that of repair k r .The support of this work by an Australian Research Council Grant (DP1093827) awarded to W. S. C., the Joint Funds of the National Natural Science Foundation of China Grant (No. U1203283) and a National Key Technology R and D Program of China Grant (2007BAD44B07) to Z. W. F., and a Knowledge Innovation Program of the Chinese Academy of Sciences grant (KZCX2- XB3-09-02) to D. -Y. F. is gratefully acknowledged

Two distinct strategies of cotton and soybean differing in leaf movement to perform photosynthesis under drought in the field

This paper reports an experimental test of the hypothesis that cotton and soybean differing in leaf movement have distinct strategies to perform photosynthesis under drought. Cotton and soybean were exposed to two water regimes: drought stressed and well watered. Drought-stressed cotton and soybean had lower maximum CO2 assimilation rates than well-watered (control) plants. Drought reduced the light saturation point and photorespiration of both species – especially in soybean. Area-based leaf nitrogen decreased in drought-stressed soybean but increased in drought-stressed cotton. Drought decreased PSII quantum yield (FPSII) in soybean leaves, but increased FPSII in cotton leaves. Drought induced an increase in light absorbed by the PSII antennae that is dissipated thermally via DpH- and xanthophylls-regulated processes in soybean leaves, but a decrease in cotton leaves. Soybean leaves appeared to have greater cyclic electron flow (CEF) around PSI than cotton leaves and drought further increased CEF in soybean leaves. In contrast, CEF slightly decreased in cotton under drought. These results suggest that the difference in leaf movement between cotton and soybean leaves gives rise to different strategies to perform photosynthesis and to contrasting photoprotective mechanisms for utilisation or dissipation of excess light energy. We suggest that soybean preferentially uses light-regulated non-photochemical energy dissipation, which may have been enhanced by the higher CEF in drought-stressed leaves. In contrast, cotton appears to rely on enhanced electron transport flux for light energy utilisation under drought, for example, in enhanced nitrogen assimilation

Over-expression of the CHS gene enhances resistance of Arabidopsis leaves to high light

Previous studies have suggested that high light (HL) stress causes photoinhibition in plants, while anthocyanins could protect the photosynthetic apparatus against photoinhibition. However, the photoprotection mechanism of anthocyanins is still ambiguous. We studied physiological responses and molecular changes for CHS-overexpression lines (CHS1, CHS2, CHS3), Arabidopsis thaliana ecotype Columbia (Col), and T-DNA insertion lines of CHS (tt4) under HL (200 μmol m−2 s−1) to explore the photoprotection mechanism of anthocyanins. The results showed that HL induced anthocyanin synthesis and accumulation. The leaves of CHS-overexpression lines turned reddest and the genes, including CHS, DFR, ANS, were expressed at highest levels. Thus, the CHS-overexpression lines maintained the highest photosynthetic capacity and suffered the least damage from HL of the three phenotypes. However, the CHS enzyme and anthocyanins were undetectable in tt4 during the experiment. Correspondingly, chlorophyll fluorescence parameters of tt4 declined greatly. The photosynthetic apparatus and cell membranes were also impaired dramatically. The physiological characteristics of Col were compared between CHS-overexpression lines and tt4. Together, the results suggest that over-expression of CHS gene enhances HL resistance by synthesizing more anthocyanins, that anthocyanins enhance the adaptability of plants to HL and that they maintain photosynthetic capacity via both antioxidation and attenuation of light.This work was funded by the National Key R&D Program of China (2017YFC1200105) and Guangdong Province Natural Science Foundation (2017A030313167, 2015A030311023). The study was also supported by the National Natural Science Foundation of China (31570398), Science and Technology Program of Guangzhou (20170701257) and Yang Cheng Scholar Program (10A040G)

Cyclic electron flow and light partitioning between the two photosystems in leaves of plants with different functional types

Cyclic electron flow (CEF) around photosystem I (PSI) is essential for generating additional ATP and enhancing efficient photosynthesis. Accurate estimation of CEF requires knowledge of the fractions of absorbed light by PSI (fI) and PSII (fII), which are only known for a few model species such as spinach. No measures of fI are available for C4 grasses under different irradiances. We developed a new method to estimate (1) fII in vivo by concurrently measuring linear electron flux through both photosystems (LEFO2) in leaf using membrane inlet mass spectrometry (MIMS) and total electron flux through PSII (ETR2) using chlorophyll fluorescence by a Dual-PAM at low light and (2) CEF as ETR1—LEFO2. For a C3 grass, fI was 0.5 and 0.4 under control (high light) and shade conditions, respectively. C4 species belonging to NADP-ME and NAD-ME subtypes had fI of 0.6 and PCK subtype had 0.5 under control. All shade-grown C4 species had fI of 0.6 except for NADP-ME grass which had 0.7. It was also observed that fI ranged between 0.3 and 0.5 for gymnosperm, liverwort and fern species. CEF increased with irradiance and was induced at lower irradiances in C4 grasses and fern relative to other species. CEF was greater in shade-grown plants relative to control plants except for C4 NADP-ME species. Our study reveals a range of CEF and fI values in different plant functional groups. This variation must be taken into account for improved photosynthetic calculations and modelling.JVS gratefully acknowledges the award of a Higher Degree Research Scholarship funded through the Centre of Excellence for Translational Photosynthesis and Western Sydney Universit

Mechanism of Photodamage of the Oxygen Evolving Mn Cluster of Photosystem II by Excessive Light Energy

Photodamage to Photosystem II (PSII) has been attributed either to excessive excitation of photosynthetic pigments or by direct of light absorption by Mn4CaO5 cluster. Here we investigated the time course of PSII photodamage and release of Mn in PSII-enriched membranes under high light illumination at 460nm and 660nm. We found that the loss of PSII activity, assayed by chlorophyll fuorescence, is faster than release of Mn from the Mn4CaO5 cluster, assayed by EPR. Loss of PSII activity and Mn release was slower during illumination in the presence of exogenous electron acceptors. Recovery of PSII activity was observed, after 30min of addition of electron donor post illumination. The same behavior was observed under 460 and 660nm illumination, suggesting stronger correlation between excessive excitation and photodamage compared to direct light absorption by the cluster. A unifed model of PSII photodamage that takes into account present and previous literature reports is presented.A.Z., M.H.C., W.C. acknowledges Australian Research Council - Discovery grant DP12010087

Antisense reductions in the PsbO protein of photosystem II leads to decreased quantum yield but similar maximal photosynthetic rates

Photosystem (PS) II is the multisubunit complex which uses light energy to split water, providing the reducing equivalents needed for photosynthesis. The complex is susceptible to damage from environmental stresses such as excess excitation energy and high temperature. This research investigated the in vivo photosynthetic consequences of impairments to PSII in Arabidopsis thaliana (ecotype Columbia) expressing an antisense construct to the PsbO proteins of PSII. Transgenic lines were obtained with between 25 and 60% of wild-type (WT) total PsbO protein content, with the PsbO1 isoform being more strongly reduced than PsbO2. These changes coincided with a decrease in functional PSII content. Low PsbO (less than 50% WT) plants grew more slowly and had lower chlorophyll content per leaf area. There was no change in content per unit area of cytochrome b6f, ATP synthase, or Rubisco, whereas PSI decreased in proportion to the reduction in chlorophyll content. The irradiance response of photosynthetic oxygen evolution showed that low PsbO plants had a reduced quantum yield, but matched the oxygen evolution rates of WT plants at saturating irradiance. It is suggested that these plants had a smaller pool of PSII centres, which are inefficiently connected to antenna pigments resulting in reduced photochemical efficiency.This work was supported by an Australian Postgraduate Award to SAD, the Australian Research Council Centre of Excellence in Plant Energy Biology (MRB), and grants from the Australian Research Council (WSC)