Heterotic growth of hybrids of Arabidopsis thaliana is enhanced by elevated atmospheric CO2.

PREMISE: With the global atmospheric CO2 concentration on the rise, developing crops that can thrive in elevated CO2 has become paramount. We investigated the potential of hybridization as a strategy for creating crops with improved growth in predicted elevated atmospheric CO2. METHODS: We grew parent accessions and their F1 hybrids of Arabidopsis thaliana in ambient and elevated atmospheric CO2 and analyzed numerous growth traits to assess their productivity and underlying mechanisms. RESULTS: The heterotic increase in total dry mass, relative growth rate and leaf net assimilation rate was significantly greater in elevated CO2 than in ambient CO2. The CO2 response of net assimilation rate was positively correlated with the CO2 response of leaf nitrogen productivity and with that of leaf traits such as leaf size and thickness, suggesting that hybridization-induced changes in leaf traits greatly affected the improved performance in elevated CO2. CONCLUSIONS: Vegetative growth of hybrids seems to be enhanced in elevated CO2 due to improved photosynthetic nitrogen-use efficiency compared with parents. The results suggest that hybrid crops should be well-suited for future conditions, but hybrid weeds may also be more competitive.

Cost-benefit analysis of mesophyll conductance: diversities of anatomical, biochemical and environmental determinants.

BACKGROUND: Plants invest photosynthates in construction and maintenance of their structures and functions. Such investments are considered costs. These costs are recovered by the CO2 assimilation rate (A) in the leaves, and thus A is regarded as the immediate, short-term benefit. In photosynthesizing leaves, CO2 diffusion from the air to the carboxylation site is hindered by several structural and biochemical barriers. CO2 diffusion from the intercellular air space to the chloroplast stroma is obstructed by the mesophyll resistance. The inverses is the mesophyll conductance (gm). Whether various plants realize an optimal gm, and how much investment is needed for a relevant gm, remain unsolved. SCOPE: This review examines relationships among leaf construction costs (CC), leaf maintenance costs (MC) and gm in various plants under diverse growth conditions. Through a literature survey, we demonstrate a strong linear relationship between leaf mass per area (LMA) and leaf CC. The overall correlation of CC vs. gm across plant phylogenetic groups is weak, but significant trends are evident within specific groups and/or environments. Investment in CC is necessary for an increase in LMA and mesophyll cell surface area (Smes). This allows the leaf to accommodate more chloroplasts, thus increasing A. However, increases in LMA and/or Smes often accompany other changes, such as cell wall thickening, which diminishes gm. Such factors that make the correlations of CC and gm elusive are identified. CONCLUSIONS: For evaluation of the contribution of gm to recover CC, leaf life span is the key factor. The estimation of MC in relation to gm, especially in terms of costs required to regulate aquaporins, could be essential for efficient control of gm over the short term. Over the long term, costs are mainly reflected in CC, while benefits also include ultimate fitness attributes in terms of integrated carbon gain over the life of a leaf, plant survival and reproductive output.

Mixed population hypothesis of the active and inactive PSII complexes opens a new door for photoinhibition and fluorescence studies: an ecophysiological perspective.

The current hypotheses for the mechanisms of photosystem II (PSII) photodamage in vivo remain split on the primary damage site. However, most researchers have considered that PSII is inhibited by a sole mechanism and that the photoinhibited PSII consists of one population. In this perspective, we propose 'the mixed population hypothesis', in which there are four PSII populations: PSII with active/inactive Mn4 CaO5 oxygen-evolving complex respectively with functional/damaged primary quinone (QA ) reduction activity. This hypothesis provides a new insight into not only the PSII photoinhibition/photoprotection studies but also the repair process. We discuss our new data implying that the repair rate differs in the respective PSII populations.

Enhanced growth rate under elevated CO2 conditions was observed for transgenic lines of genes identified by intraspecific variation analyses in Arabidopsis thaliana.

KEY MESSAGE: Using the whole genome and growth data of Arabidopsis thaliana ecotypes, we identified two genes associated with enhancement of the growth rate in response to elevated CO2 conditions. Improving plant growth under elevated CO2 conditions may contribute to enhanced agricultural yield under future global climate change. In this study, we examined the genes implicated in the enhancement of growth rates under elevated CO2 conditions by analyzing the growth rates of Arabidopsis thaliana ecotypes originating from various latitudes and altitudes throughout the world. We also performed a genome-wide association study and a transcriptome study to identify single nucleic polymorphisms that were correlated with the relative growth rate (RGR) under elevated CO2 conditions or with CO2 response of RGR. We then selected 43 candidate genes and generated their overexpression and/or RNA interference (RNAi) transgenic mutants for screening. After screening, we have found that RNAi lines of AT3G4000 and AT5G50900 showed significantly higher growth rates under the elevated CO2 condition. As per our findings, we conclude that natural variation includes genetic variation associated with the enhancement of plant productivity under elevated CO2 conditions.

A new method for separate evaluation of PSII with inactive oxygen evolving complex and active D1 by the pulse-amplitude modulated chlorophyll fluorometry.

A method that separately quantifies the PSII with inactive oxygen-evolving complex (OEC) and active D1 retaining the primary quinone acceptor (QA )-reducing activity from the PSII with damaged D1 in the leaf was developed using PAM fluorometry. It is necessary to fully reduce QA to obtain F m , the maximum fluorescence. However, QA in PSII with inactive OEC and active D1 would not be fully reduced by a saturating flash. We used the acceptor-side inhibitor DCMU to fully reduce QA . Leaves of cucumber (Cucumis sativus L.) were chilled at 4°C in dark or illuminated with UV-A to selectively inactivate OEC. After these treatments, F v /F m , the maximum quantum yield, in the leaves vacuum-infiltrated with DCMU were greater than those in water-infiltrated leaves. In contrast, when the leaves were illuminated by red light to photodamage D1, F v /F m did not differ between DCMU- and water-infiltrated leaves. These results indicate relevance of the present evaluation of the fraction of PSII with inactive OEC and active D1. Several examinations in the laboratory and glasshouse showed that PSII with inactive OEC and active D1 was only rarely observed. The present simple method would serve as a useful tool to clarify the details of the PSII photoinhibition.

Wah Soon Chow, a teacher, a friend and a colleague.

To finish this special issue, some friends, colleagues and students of Prof. Chow (Emeritus Professor, the Research School of Biology, the Australian National University) have written small tributes to acknowledge not only his eminent career but to describe his wonderful personality.

The effect of different spectral light quality on the photoinhibition of Photosystem I in intact leaves.

Light energy causes damage to Photosystem I (PSI) and Photosystem II (PSII). The majority of the previous photoinhibition studies have been conducted with PSII, which shows much larger photoinhibition than PSI; therefore, relatively little is known about the mechanism of PSI photoinhibition so far. A previous report showed that the photoinhibition action spectrum measured with PSI activity of isolated thylakoid is similar to the absorption spectrum of chlorophyll. However, it is known that the extent of PSI photoinhibition is much smaller in vivo compared to in vitro. It is also possible that the different extent of PSII photoinhibition, caused by different spectral light qualities, can affect the photoinhibition of PSI in vivo because PSI receives electrons from PSII. In the present research, to study the effect of light quality and the effect of the extent of PSII photoinhibition on the PSI photoinhibition in vivo, intact leaves were photoinhibited under four different light qualities. The rate coefficient of PSI photoinhibition was significantly higher in blue and red light compared to white light. The rate of PSI photoinhibition at the same photon-exposure was the largest in blue and red light and followed by white and green light. These results support the notion that light absorption by chlorophyll is responsible for the PSI photoinhibition, even in intact leaves. The variation among light colors in the relationships between the extent of photoinhibition of PSII and that of PSI indicate that PSI and PSII are independently photoinhibited with different mechanisms in the early stage of in vivo photoinhibition.

Functional shifts in leaves of woody invaders of deciduous forests between their home and away ranges.

Temperate forests are widely invaded by shade-tolerant shrubs and trees, including those of Eastern North America (ENA). However, it remains unknown whether these invaders are 'preadapted' for success in their new ranges due to unique aspects of their evolutionary history or whether selection due to enemy release or other postintroduction processes have driven rapid evolution in the invaded range. We sampled leaf traits of populations of woody understory invaders across light gradients in their native range in Japan and in their invaded ENA range to examine potential phenotypic shifts related to carbon gain and nitrogen use between ranges. We also measured leaf traits in three co-occurring ENA native shrub species. In their invaded range, invaders invested significantly less in leaf chlorophyll content (both per unit leaf mass and area) compared with native range populations of the same species, yet maintained similar rates of photosynthesis in low light. In addition, compared with ENA natives, ENA invaders displayed greater trait variation in response to increasing light availability (forest edges, gaps), giving them a potential advantage over ENA natives in a variety of light conditions. We conclude that, for this group of species, newly evolved phenotypes in the invaded range are more important than preadaptation for their success as shade-tolerant forest invaders.

Dependence of functional traits related to growth rates and their CO2 response on multiple habitat climate factors across Arabidopsis thaliana populations.

The values of many plant traits are often different even within a species as a result of local adaptation. Here, we studied how multiple climate variables influence trait values in Arabidopsis thaliana grown under common conditions. We examined 9 climate variables and 29 traits related to vegetative growth rate in 44 global A. thaliana accessions grown at ambient or elevated CO2 concentration ([CO2]) and applied a multiple regression analysis. We found that genetic variations in the traits related to growth rates were associated with various climate variables. At ambient [CO2], plant size was positively correlated with precipitation in the original habitat. This may be a result of larger biomass investment in roots at the initial stage in plants adapting to a lower precipitation. Stomatal conductance and photosynthetic nitrogen use efficiency were negatively correlated with vapor pressure deficit, probably as a result of the trade-off between photosynthetic water- and nitrogen-use efficiency. These results suggest that precipitation and air humidity influence belowground and aboveground traits, respectively. Elevated [CO2] altered climate dependences in some of the studied traits. The CO2 response of relative growth rate was negatively correlated with altitude, indicating that plants inhabiting a higher altitude have less plasticity to changing [CO2]. These results are useful not only for understanding evolutionary process but also to predict the plant species that are favored under future global change.

Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING.

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

The effect of interspecific variation in photosynthetic plasticity on 4-year growth rate and 8-year survival of understorey tree seedlings in response to gap formations in a cool-temperate deciduous forest.

Gap formation increases the light intensity in the forest understorey. The growth responses of seedlings to the increase in light availability show interspecific variation, which is considered to promote biodiversity in forests. At the leaf level, some species increase their photosynthetic capacity in response to gap formation, whereas others do not. Here we address the question of whether the interspecific difference in the photosynthetic response results in the interspecific variation in the growth response. If so, the interspecific difference in photosynthetic response would also contribute to species coexistence in forests. We also address the further relevant question of why some species do not increase their photosynthetic capacity. We assumed that some cost of photosynthetic plasticity may constrain acquisition of the plasticity in some species, and hypothesized that species with larger photosynthetic plasticity exhibit better growth after gap formation and lower survivorship in the shade understorey of a cool-temperate deciduous forest. We created gaps by felling canopy trees and studied the relationship between the photosynthetic response and the subsequent growth rate of seedlings. Naturally growing seedlings of six deciduous woody species were used and their mortality was examined for 8 years. The light-saturated rate of photosynthesis (Pmax) and the relative growth rate (RGR) of the seedlings of all study species increased at gap plots. The extent of these increases varied among the species. The stimulation of RGR over 4 years after gap formation was strongly correlated with change in photosynthetic capacity of newly expanded leaves. The increase in RGR and Pmax correlated with the 8-year mortality at control plots. These results suggest a trade-off between photosynthetic plasticity and the understorey shade tolerance. Gap-demanding species may acquire photosynthetic plasticity, sacrificing shade tolerances, whereas gap-independent species may acquire shade tolerances, sacrificing photosynthetic plasticity. This strategic difference among species would contribute to species coexistence in cool-temperate deciduous forests.

Obstacles in the quantification of the cyclic electron flux around Photosystem I in leaves of C3 plants.

Sixty years ago Arnon and co-workers discovered photophosphorylation driven by a cyclic electron flux (CEF) around Photosystem I. Since then understanding the physiological roles and the regulation of CEF has progressed, mainly via genetic approaches. One basic problem remains, however: quantifying CEF in the absence of a net product. Quantification of CEF under physiological conditions is a crucial prerequisite for investigating the physiological roles of CEF. Here we summarize current progress in methods of CEF quantification in leaves and, in some cases, in isolated thylakoids, of C3 plants. Evidently, all present methods have their own shortcomings. We conclude that to quantify CEF in vivo, the best way currently is to measure the electron flux through PS I (ETR1) and that through PS II and PS I in series (ETR2) for the whole leaf tissue under identical conditions. The difference between ETR1 and ETR2 is an upper estimate of CEF, mainly consisting, in C3 plants, of a major PGR5-PGRL1-dependent CEF component and a minor chloroplast NDH-dependent component, where PGR5 stands for Proton Gradient Regulation 5 protein, PGRL1 for PGR5-like photosynthesis phenotype 1, and NDH for Chloroplast NADH dehydrogenase-like complex. These two CEF components can be separated by the use of antimycin A to inhibit the former (major) component. Membrane inlet mass spectrometry utilizing stable oxygen isotopes provides a reliable estimation of ETR2, whilst ETR1 can be estimated from a method based on the photochemical yield of PS I, Y(I). However, some issues for the recommended method remain unresolved.

Which plant trait explains the variations in relative growth rate and its response to elevated carbon dioxide concentration among Arabidopsis thaliana ecotypes derived from a variety of habitats?

Elevated atmospheric carbon dioxide (CO2) concentration ([CO2]) enhances plant growth, but this enhancement varies considerably. It is still uncertain which plant traits are quantitatively related to the variation in plant growth. To identify the traits responsible, we developed a growth analysis model that included primary parameters associated with morphology, nitrogen (N) use, and leaf and root activities. We analysed the vegetative growth of 44 ecotypes of Arabidopsis thaliana L. grown at ambient and elevated [CO2] (800 µmol mol(-1)). The 44 ecotypes were selected such that they were derived from various altitudes and latitudes. Relative growth rate (RGR; growth rate per unit plant mass) and its response to [CO2] varied by 1.5- and 1.7-fold among ecotypes, respectively. The variation in RGR at both [CO2]s was mainly explained by the variation in leaf N productivity (LNP; growth rate per leaf N),which was strongly related to photosynthetic N use efficiency (PNUE). The variation in the response of RGR to [CO2] was also explained by the variation in the response of LNP to [CO2]. Genomic analyses indicated that there was no phylogenetic constraint on inter-ecotype variation in the CO2 response of RGR or LNP. We conclude that the significant variation in plant growth and its response to [CO2] among ecotypes reflects the variation in N use for photosynthesis among ecotypes, and that the response of PNUE to CO2 is an important target for predicting and/or breeding plants that have high growth rates at elevated [CO2].

Cotton bracts are adapted to a microenvironment of concentrated CO2 produced by rapid fruit respiration.

BACKGROUND AND AIMS: Elucidation of the mechanisms by which plants adapt to elevated CO2 is needed; however, most studies of the mechanisms investigated the response of plants adapted to current atmospheric CO2. The rapid respiration rate of cotton (Gossypium hirsutum) fruits (bolls) produces a concentrated CO2 microenvironment around the bolls and bracts. It has been observed that the intercellular CO2 concentration of a whole fruit (bract and boll) ranges from 500 to 1300 µmol mol(-1) depending on the irradiance, even in ambient air. Arguably, this CO2 microenvironment has existed for at least 1·1 million years since the appearance of tetraploid cotton. Therefore, it was hypothesized that the mechanisms by which cotton bracts have adapted to elevated CO2 will indicate how plants will adapt to future increased atmospheric CO2 concentration. Specifically, it is hypothesized that with elevated CO2 the capacity to regenerate ribulose-1,5-bisphosphate (RuBP) will increase relative to RuBP carboxylation. METHODS: To test this hypothesis, the morphological and physiological traits of bracts and leaves of cotton were measured, including stomatal density, gas exchange and protein contents. KEY RESULTS: Compared with leaves, bracts showed significantly lower stomatal conductance which resulted in a significantly higher water use efficiency. Both gas exchange and protein content showed a significantly greater RuBP regeneration/RuBP carboxylation capacity ratio (Jmax/Vcmax) in bracts than in leaves. CONCLUSIONS: These results agree with the theoretical prediction that adaptation of photosynthesis to elevated CO2 requires increased RuBP regeneration. Cotton bracts are readily available material for studying adaption to elevated CO2.

Estimation of the steady-state cyclic electron flux around PSI in spinach leaf discs in white light, CO2-enriched air and other varied conditions.

Cyclic electron flux (CEF) around PSI is essential for efficient photosynthesis and aids photoprotection, especially in stressful conditions, but the difficulty in quantifying CEF is non-trivial. The total electron flux through PSI (ETR1) and the linear electron flux (LEFO2) through both photosystems in spinach leaf discs were estimated from the photochemical yield of PSI and the gross oxygen evolution rate, respectively, in CO2-enriched air. ΔFlux=ETR1 - LEFO2 is an upper estimate of CEF. Infiltration of leaf discs with 150µM antimycin A did not affect LEFO2, but decreased ΔFlux 10-fold. ΔFlux was practically negligible below 350µmolphotonsm-2s-1, but increased linearly above it. The following results were obtained at 980µmolphotonsm-2s-1. ΔFlux increased 3-fold as the temperature increased from 5°C to 40°C. It did not decline at high temperature, even when LEFO2 decreased. ΔFlux increased by 80% as the relative water content of leaf discs decreased from 100 to 40%, when LEFO2 decreased 2-fold. The method of using ΔFlux as a non-intrusive upper estimate of steady-state CEF in leaf tissue appears reasonable when photorespiration is suppressed.

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 O(2) yield per single-turnover flash in CO(2)-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O(2) molecule after four flashes. However, the gross O(2) yield per single-turnover flash (multiplied by four) could over-estimate 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.

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.

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 wall in cotton by measuring their time-course of surface area development, O(2) 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 O(2) evolution capacity per area and photosynthetic enzyme activity than leaves at the late growth stage. The total capacity for O(2) evolution of stalks and bolls (bracts plus capsule wall) was 12.7 and 23.7% (total ca. 36.4%), respectively, as estimated by multiplying their surface area by their O(2) 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.

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 of whether photon absorption by chlorophyll or by manganese (Mn) is the primary cause of photoinhibition, as 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 microfiber 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 the deeper tissue, which correlated with the chlorophyll and the Mn absorption spectrums, respectively. These results cannot be explained by either hypothesis alone. • These data strongly suggest that both the excess-energy and the two-step mechanisms occur in photoinhibition, and fluorometers with red or blue measuring light give overestimated or underestimated F(v)/F(m) values of photoinhibited leaves compared with the whole tissue average, respectively; that is, they measured deeper or shallower leaf tissue, respectively.

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.