Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b6 f.

Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.

Probing functional and optical cross-sections of PSII in leaves during state transitions using fast repetition rate light induced fluorescence transients.

Plants adjust the relative sizes of PSII and PSI antennae in response to the spectral composition of weak light favouring either photosystem by processes known as state transitions (ST), attributed to a discrete antenna migration involving phosphorylation of light-harvesting chlorophyll-protein complexes in PSII. Here for the first time we monitored the extent and dynamics of ST in leaves from estimates of optical absorption cross-section (relative PSII antenna size; aPSII). These estimates were obtained from in situ measurements of functional absorption cross-section (σPSII) and maximum photochemical efficiency of PSII (φPSII); i.e. aPSII = σPSII/φPSII (Kolber et al. 1998) and other parameters from a light induced fluorescence transient (LIFT) device (Osmond et al. 2017). The fast repetition rate (FRR) QA flash protocol of this instrument monitors chlorophyll fluorescence yields with reduced QA irrespective of the redox state of plastoquinone (PQ), as well as during strong ~1 s white light pulses that fully reduce the PQ pool. Fitting this transient with the FRR model monitors kinetics of PSII → PQ, PQ → PSI, and the redox state of the PQ pool in the 'PQ pool control loop' that underpins ST, with a time resolution of a few seconds. All LIFT/FRR criteria confirmed the absence of ST in antenna mutant chlorina-f2 of barley and asLhcb2-12 of Arabidopsis, as well as STN7 kinase mutants stn7 and stn7/8. In contrast, wild-type barley and Arabidopsis genotypes Col, npq1, npq4, OEpsbs, pgr5 bkg and pgr5, showed normal ST. However, the extent of ST (and by implication the size of the phosphorylated LHCII pool participating in ST) deduced from changes in a'PSII and other parameters with reduced QA range up to 35%. Estimates from strong WL pulses in the same assay were only ~10%. The larger estimates of ST from the QA flash are discussed in the context of contemporary dynamic structural models of ST involving formation and participation of PSII and PSI megacomplexes in an 'energetically connected lake' of phosphorylated LHCII trimers (Grieco et al. 2015). Despite the absence of ST, asLhcb2-12 displays normal wild-type modulation of electron transport rate (ETR) and the PQ pool during ST assays, reflecting compensatory changes in antenna LHCIIs in this genotype. Impaired LHCII phosphorylation in stn7 and stn7/8 accelerates ETR from PSII →PQ, over-reducing the PQ pool and abolishing the yield difference between the QA flash and WL pulse, with implications for photochemical and thermal phases of the O-J-I-P transient.

Remote monitoring of dynamic canopy photosynthesis with high time resolution light-induced fluorescence transients.

Understanding the net photosynthesis of plant canopies requires quantifying photosynthesis in challenging environments, principally due to the variable light intensities and qualities generated by sunlight interactions with clouds and surrounding foliage. The dynamics of sunflecks and rates of change in light intensity at the beginning and end of sustained light (SL) events makes photosynthetic measurements difficult, especially when dealing with less accessible parts of plant foliage. High time resolved photosynthetic monitoring from pulse amplitude modulated (PAM) fluorometers has limited applicability due to the invasive nature of frequently applied saturating flashes. An alternative approach used here provides remote (<5 m), high time resolution (10 s), PAM equivalent but minimally invasive measurements of photosynthetic parameters. We assessed the efficacy of the QA flash protocol from the Light-Induced Fluorescence Transient (LIFT) technique for monitoring photosynthesis in mature outer canopy leaves of potted Persea americana Mill. cv. Haas (Avocado) trees in a semi-controlled environment and outdoors. Initially we established that LIFT measurements were leaf angle independent between ±40° from perpendicular and moreover, that estimates of 685 nm reflectance (R685) from leaves of similar chlorophyll content provide a species dependent, but reasonable proxy for incident light intensity. Photosynthetic responses during brief light events (≤10 min), and the initial stages of SL events, showed similar declines in the quantum yield of photosystem II (ΦII) with large transient increases in 'constitutive loss processes' (ΦNO) prior to dissipation of excitation by non-photochemical quenching (ΦNPQ). Our results demonstrate the capacity of LIFT to monitor photosynthesis at a distance during highly dynamic light conditions that potentially may improve models of canopy photosynthesis and estimates of plant productivity. For example, generalized additive modelling performed on the 85 dynamic light events monitored identified negative relationships between light event length and ∆ΦII and ∆electron transport rate using either ∆photosynthetically active radiation or ∆R685 as indicators of leaf irradiance.

Relative functional and optical absorption cross-sections of PSII and other photosynthetic parameters monitored in situ, at a distance with a time resolution of a few seconds, using a prototype light induced fluorescence transient (LIFT) device.

The prototype light-induced fluorescence transient (LIFT) instrument provides continuous, minimally intrusive, high time resolution (~2s) assessment of photosynthetic performance in terrestrial plants from up to 2m. It induces a chlorophyll fluorescence transient by a series of short flashes in a saturation sequence (180 ~1µs flashlets in <380µs) to achieve near-full reduction of the primary acceptor QA, followed by a relaxation sequence (RQA; 90 flashlets at exponentially increasing intervals over ~30ms) to observe kinetics of QA re-oxidation. When fitted by the fast repetition rate (FRR) model (Kolber et al. 1998) the QA flash of LIFT/FRR gives smaller values for FmQA from dark adapted leaves than FmPAM from pulse amplitude modulated (PAM) assays. The ratio FmQA/FmPAM resembles the ratio of fluorescence yield at the J/P phases of the classical O-J-I-P transient and we conclude that the difference simply is due to the levels of PQ pool reduction induced by the two techniques. In a strong PAM-analogous WL pulse in the dark monitored by the QA flash of LIFT/FRR φPSIIWL ≈ φPSIIPAM. The QA flash also tracks PQ pool reduction as well as the associated responses of ETR QA → PQ and PQ → PSI, the relative functional (σPSII) and optical absorption (aPSII) cross-sections of PSII in situ with a time resolution of ~2s as they relax after the pulse. It is impractical to deliver strong WL pulses at a distance in the field but a longer PQ flash from LIFT/FRR also achieves full reduction of PQ pool and delivers φPSIIPQ ≈ φPSIIPAM to obtain PAM-equivalent estimates of ETR and NPQ at a distance. In situ values of σPSII and aPSII from the QA flash with smaller antenna barley (chlorina-f2) and Arabidopsis mutants (asLhcb2-12, ch1-3 Lhcb5) are proportionally similar to those previously reported from in vitro assays. These direct measurements are further validated by changes in antenna size in response to growth irradiance. We illustrate how the QA flash facilitates our understanding of photosynthetic regulation during sun flecks in natural environments at a distance, with a time resolution of a few seconds.

Our eclectic adventures in the slower eras of photosynthesis: from New England Down Under to biosphere 2 and beyond.

This is a tale of a career in plant physiological ecology that enjoyed the freedom to address photosynthetic physiology and biochemistry in leaves of plants from diverse environments. It was supported by block funding (now sadly a thing of the past) for research at the Australian National University, by grants during appointments in the United States and in Germany, and by Columbia University. It became a "career experiment" in which long-term, high-trust support for curiosity-driven plant biology in Australia, and at times in the United States, led to surprisingly innovative results. Although the rich diversity of short-term competitive grant opportunities in the United States sustained ongoing research, it proved difficult to mobilize support for more risky long-term projects. A decade after the closure of the Biosphere 2 Laboratory, this article highlights the achievements of colleagues in experimental climate change research from 1998 to 2003.

Integrating transient heterogeneity of non-photochemical quenching in shade-grown heterobaric leaves of avocado (Persea americana L.): responses to CO2 concentration, stomatal occlusion, dehydration and relative humidity.

Long-lived shade leaves of avocado had extremely low rates of photosynthesis. Gas exchange measurements of photosynthesis were of limited use, so we resorted to Chl fluorescence imaging (CFI) and spot measurements to evaluate photosynthetic electron transport rates (ETRs) and non-photochemical quenching (NPQ). Imaging revealed a remarkable transient heterogeneity of NPQ during photosynthetic induction in these hypostomatous, heterobaric leaves, but was adequately integrated by spot measurements, despite long-lasting artifacts from repeated saturating flashes during assays. Major veins (mid-vein, first- and second-order veins) defined areas of more static large-scale heterogeneous NPQ, with more dynamic small-scale heterogeneity most strongly expressed in mesophyll cells between third- and fourth-order veins. Both responded to external CO2 concentration ([CO2]), occlusion of stomata with Vaseline™, leaf dehydration and relative humidity (RH). We interpreted these responses in terms of independent behavior of stomata in adjacent areoles that was largely expressed through CO2-limited photosynthesis. Heterogeneity was most pronounced and prolonged in the absence of net CO2 fixation in 100 p.p.m. [CO2] when respiratory and photorespiratory CO2 cycling constrained the inferred ETR to ~75% of values in 400 or 700 p.p.m. [CO2]. Likewise, sustained higher NPQ under Vaseline™, after dehydration or at low RH, also restricted ETR to ~75% of control values. Low NPQ in chloroplast-containing cells adjacent to major veins but remote from stomata suggested internal sources of high [CO2] in these tissues.

From ecophysiology to phenomics: some implications of photoprotection and shade-sun acclimation in situ for dynamics of thylakoids in vitro.

Half a century of research into the physiology and biochemistry of sun-shade acclimation in diverse plants has provided reality checks for contemporary understanding of thylakoid membrane dynamics. This paper reviews recent insights into photosynthetic efficiency and photoprotection from studies of two xanthophyll cycles in old shade leaves from the inner canopy of the tropical trees Inga sapindoides and Persea americana (avocado). It then presents new physiological data from avocado on the time frames of the slow coordinated photosynthetic development of sink leaves in sunlight and on the slow renovation of photosynthetic properties in old leaves during sun to shade and shade to sun acclimation. In so doing, it grapples with issues in vivo that seem relevant to our increasingly sophisticated understanding of ΔpH-dependent, xanthophyll-pigment-stabilized non-photochemical quenching in the antenna of PSII in thylakoid membranes in vitro.

Canopy conundrums: building on the Biosphere 2 experience to scale measurements of inner and outer canopy photoprotection from the leaf to the landscape.

Recognising that plant leaves are the fundamental productive units of terrestrial vegetation and the complexity of different environments in which they must function, this review considers a few of the ways in which these functions may be measured and potentially scaled to the canopy. Although canopy photosynthetic productivity is clearly the sum of all leaves in the canopy, we focus on the quest for 'economical insights' from measurements that might facilitate integration of leaf photosynthetic activities into canopy performance, to better inform modelling based on the 'insights of economics'. It is focussed on the reversible downregulation of photosynthetic efficiency in response to light environment and stress and summarises various xanthophyll-independent and dependent forms of photoprotection within the inner and outer canopy of woody plants. Two main themes are developed. First, we review experiments showing the retention of leaves that grow old in the shade may involve more than the 'payback times' required to recover the costs of their construction and maintenance. In some cases at least, retention of these leaves may reflect selection for distinctive properties that contribute to canopy photosynthesis through utilisation of sun flecks or provide 'back up' capacity following damage to the outer canopy. Second, we report experiments offering hope that remote sensing of photosynthetic properties in the outer canopy (using chlorophyll fluorescence and spectral reflectance technologies) may overcome problems of access and provide integrated measurements of these properties in the canopy as a whole. Finding appropriate tools to scale photosynthesis from the leaf to the landscape still presents a challenge but this synthesis identifies some measurements and criteria in the laboratory and the field that improve our understanding of inner and outer canopy processes.

Curiosity and context revisited: crassulacean acid metabolism in the Anthropocene.

Having gained some understanding of the consequences of the CO(2)-concentrating mechanisms in crassulacean acid metabolism (CAM) that internalize the photosynthetic environment of the Cretaceous on a daily basis, it may be time to consider potential long-term effects of the planetary CO(2)-concentrating mechanism on growth and ecology of these plants in the Anthropocene. This paper emphasizes our limited understanding of the carbohydrate economy of CAM in relation to growth processes and briefly reviews recent studies of the diel cycles of growth in these plants. An inadvertent long-term, regional-scale experiment from the past is revisited in which an Opuntia monoculture grew to occupy >25 million hectares of farmland in central eastern Australia, producing a total biomass of about 1.5 billion tonnes in about 80 years. Although at the time it does not seem to have been recognized that this invasion involved CAM, a botanist from the University of Melbourne, Jean White-Haney emerges as a heroic pioneer in the control of the invader by poison and pioneered its biological control. The Opuntia population was expanding at 10-100 ha h(-1) when it was brought to a halt within a decade by the voracious appetite of Cactoblastis cactorum larvae. It is now known that the female parent moth of this predator detects CAM in O. stricta prior to oviposition by deploying the most sensitive CO(2) detector system yet found in the Lepidoptera. The O. stricta invasion is a dramatic demonstration of the capacity of CAM plants to attain and sustain high biomass; to sequester and retain atmospheric CO(2). In conclusion, experiments are reviewed that show stimulation of CO(2) assimilation, growth, and biomass of CAM plants by elevated atmospheric [CO(2)], and the proposition that these plants may have a role in atmospheric CO(2) sequestration is re-examined. This role may be compromised by predators such as Cactoblastis. However the moth CO(2) sensors are adapted to pre-industrial atmospheric [CO(2)] and FACE (free-air CO(2) enrichment) experiments show this exquisite system of biological control is also compromised by rising global [CO(2)] in the Anthropocene.

Diel leaf growth cycles in Clusia spp. are related to changes between C3 photosynthesis and crassulacean acid metabolism during development and during water stress.

This study reports evidence that the timing of leaf growth responds to developmental and environmental constraints in Clusia spp. We monitored diel patterns of leaf growth in the facultative C(3)-crassulacean acid metabolism (CAM) species Clusia minor and in the supposedly obligate CAM species Clusia alata using imaging methods and followed diel patterns of CO2 exchange and acidification. Developing leaves of well-watered C. minor showed a C3-like diel pattern of gas exchange and growth, with maximum relative growth rate (RGR) in the early night period. Growth slowed when water was withheld, accompanied by nocturnal CO2 exchange and the diel acid change characteristic of CAM. Maximum leaf RGR shifted from early night to early in the day when water was withheld. In well-watered C. alata, similar changes in the diel pattern of leaf growth occurred with the development of CAM during leaf ontogeny. We hypothesize that the shift in leaf growth cycle that accompanies the switch from C3 photosynthesis to CAM is mainly caused by the primary demand of CAM for substrates for nocturnal CO2 fixation and acid synthesis, thus reducing the availability of carbohydrates for leaf growth at night. Although the shift to leaf growth early in the light is presumably associated with the availability of carbohydrates, source-sink relationships and sustained diurnal acid levels in young leaves of Clusia spp. need further evaluation in relation to growth processes.

Measuring photosynthetic parameters at a distance: laser induced fluorescence transient (LIFT) method for remote measurements of photosynthesis in terrestrial vegetation.

We have developed a laser induced fluorescence transient (LIFT) technique and instrumentation to remotely measure photosynthetic properties in terrestrial vegetation at a distance of up to 50 m. The LIFT method uses a 665 nm laser to project a collimated, 100 mm diameter excitation beam onto leaves of the targeted plant. Fluorescence emission at 690 nm is collected by a 250 mm reflective telescope and processed in real time to calculate the efficiency of photosynthetic light utilization, quantum efficiency of PS II, and the kinetics of photosynthetic electron transport. Operating with peak excitation power of 125 W m-2, and duty cycle of 10-50%, the instrument conforms to laser safety regulations. The LIFT instrument is controlled via an Internet connection, allowing it to operate from remote locations or platforms. Here we describe the theoretical basis of the LIFT methodology, and demonstrate its applications in remote measurements of photosynthetic properties in the canopy of cottonwood and oak trees, and in the rosette of Arabidopsis mutants.

Slowly reversible de-epoxidation of lutein-epoxide in deep shade leaves of a tropical tree legume may 'lock-in' lutein-based photoprotection during acclimation to strong light.

The kinetics of response to strong light have been examined in deeply shaded leaves of the tropical tree legume (Inga sp.) which have extraordinarily high levels of the alpha-xanthophyll lutein-epoxide that are co-located in pigment-protein complexes of the photosynthetic apparatus with the beta-xanthophyll violaxanthin. As in other species, rapidly reversible photoprotection (measured as non-photochemical chlorophyll fluorescence quenching) is initiated within the time frame of sun-flecks (minutes), before detectable conversion of violaxanthin to antheraxanthin or zeaxanthin. Photoprotection is stabilized within hours of exposure to strong light by simultaneously engaging the reversible violaxanthin cycle and a slowly reversible conversion of lutein-epoxide to lutein. It is proposed that this lutein 'locks in' a primary mechanism of photoprotection during photoacclimation in this species, converting efficient light-harvesting antennae of the shade plant into potential excitation dissipating centres. It is hypothesized that lutein occupies sites L2 and V1 in light-harvesting chlorophyll protein complexes of photosystem II, facilitating enhanced photoprotection through the superior singlet and/or triplet chlorophyll quenching capacity of lutein.