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

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

Quantifying and monitoring functional Photosystem II and the stoichiometry of the two photosystems in leaf segments: Approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O2 yield per single-turnover flash in CO2-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O2 molecule after four flashes. However, the gross O2 yield per single-turnover flash (multiplied by four) could overestimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700? (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field

Testing trait plasticity over the range of spectral composition of sunlight in forb species differing in shade tolerance

Although sunlight is essential for plant growth and development, the relative importance of each spectral region in shaping functional traits is poorly understood, particularly in dynamic light environments such as forest ecosystems. We examined responses of 25 functional traits from groups of 11 shade-intolerant and 12 understorey shade-tolerant forb species grown outdoors under five filter treatments differing in spectral transmittance: (a) transmitting c. 95% of solar radiation (280-800 nm); (b) attenuating ultraviolet-B (UV-B); (c) attenuating all UV; (d) attenuating all UV and blue light; (e) attenuating all UV, blue and green light. Our results show that UV-B radiation mainly affected the biochemical traits but blue light mainly affected the physiological traits irrespective of functional strategy, whereas green light affected both sets of traits. This would suggest that differentiation among suites of functional trait responses proceeds according to light quality. Biomass accumulation was significantly increased by UV-A radiation (contrasting treatment [b] vs. [c]) among shade-intolerant but decreased by blue light among shade-tolerant species; green and red light affected whole-plant morphological development differently according to functional groups. Shade-tolerant species were more plastic than shade-intolerant species in response to each spectral region that we examined except for UV-B radiation. Synthesis. Our results show that differences in the spectral composition of sunlight can drive functional trait expression irrespective of total irradiance received. The different responses of functional traits between functional groups imply that shade-tolerant and intolerant species have adapted to utilize spectral cues differently in their respective light environments.Peer reviewe

Important photosynthetic contribution from the non-foliar green organs in cotton at the late growth stage

Non-foliar green organs are recognized as important carbon sources after leaves. However, the contribution of each organ to total yield has not been comprehensively studied in relation to the time-course of changes in surface area and photosynthetic activity of different organs at different growth stages. We studied the contribution of leaves, main stem, bracts and capsule walls in cotton by measuring their time-course of surface area development, O2 evolution capacity and photosynthetic enzyme activity. Because of the early senescence of leaves, non-foliar organs increased their surface area up to 38.2% of total at late growth stage. Bracts and capsule wall showed less ontogenetic decrease in O2 evolution capacity per area and photosynthetic enzyme activity than leaves at the late growth stage. The total capacity for O2 evolution of stalks and bolls (bracts plus capsule wall) was 12.7% and 23.7% (total ca. 36.4%), respectively, as estimated by m! ultiplying their surface area by their O2 evolution capacity per area. We also kept the bolls (from 15 days after anthesis) or main stem (at the early full bolling stage) in darkness for comparison with non-darkened controls. Darkening the bolls and main stem reduced the boll weight by 24.1% and 9%, respectively, and the seed weight by 35.9% and 16.3%, respectively. We conclude that non-foliar organs significantly contribute to the yield at the late growth stage

The involvement of dual mechanisms of photoinactivation of photosystem II in Capsicum annuum L. plants

For plants, light is an indispensable resource. However, it also causes a loss of photosynthetic activity associated with photoinactivation of photosystem II (PSII). In studies of the mechanism of this photoinactivation, there are two conflicting hypotheses at present. One is that excess energy received by leaves, being neither utilized by photosynthesis nor dissipated safely in non-photochemical quenching, causes the photoinactivation. The other involves a two-step mechanism in which excitation of Mn by photons is the primary cause. In the former hypothesis, photoinactivation of PSII should not occur in low light that provides little excess energy, but in the latter hypothesis it should. Therefore, we tested these two hypotheses in different irradiances. We used a system that can measure the fraction of functional PSII complexes under natural conditions and over a long period in intact leaves, which were attached to a plant treated with lincomycin taken up via the roots. The leaves were photoinactivated in low, medium or high light (30, 60 or 950 μmol m-2 s-1) with white, blue, green or red light-emitting diode arrays. Our results showed that the extent of photoinactivation per photon exposure was higher in high light than in low light, consistent with the abundance of excess energy. However, photoinactivation did occur in low light with little excess energy, and blue light caused the greatest extent of photoinactivation followed by white, green and red light in this order, an order that can be predicted from the Mn absorbance spectrum. These results suggest that both mechanisms occur in the photoinactivation process

Operation of dual mechanisms that both lead to photoinactivation of Photosystem II in leaves by visible light

Photosystem II (PS II) is photoinactivated during photosynthesis, requiring repair to maintain full function during the day. What is the mechanism(s) of the initial events that lead to photoinactivation of PS II? Two hypotheses have been put forward. The ‘excess-energy hypothesis' states that excess energy absorbed by chlorophyll (Chl), neither utilized in photosynthesis nor dissipated harmlessly in non-photochemical quenching, leads to PS II photoinactivation; the ‘Mn hypothesis' (also termed the two-step hypothesis) states that light absorption by the Mn cluster in PS II is the primary effect that leads to dissociation of Mn, followed by damage to the reaction centre by light absorption by Chl. Observations from various studies support one or the other hypothesis, but each hypothesis alone cannot explain all the observations. We propose that both mechanisms operate in the leaf, with the relative contribution from each mechanism depending on growth conditions or plant species. Indeed, in a single system, namely, the interior of a leaf, we could observe one or the other mechanism at work, depending on the location within the tissue. There is no reason to expect the two mechanisms to be mutually exclusive.This work was supported by a JSPS Research Fellowship for Young Scientists (18-8553 to R. O.); a JSPS Postdoctoral Fellowships for Research Abroad (to R. O.); an Australian Research Council (DP1093827 to W. S. C.); a China Scholarship Council Fellowship (to J. K.) and a Grant-in-Aid for Challenging Exploratory Research (21657007 to I. T.)

Recovery of photoinactivated photosystem II in leaves: retardation due to restricted mobility of photosystem II in the thylakoid membrane

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as Fv/Fm, the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither Fv/Fm nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair