Chlorophyll fluorescence is a non-invasive measurement of photosystem II (PSII) activity and is a commonly used technique in plant physiology. The sensitivity of PSII activity to abiotic and biotic factors has made this a key technique not only for understanding the photosynthetic mechanisms but also as a broader indicator of how plants respond to environmental change. This, along with low cost and ease of collecting data, has resulted in the appearance of a large array of instrument types for measurement and calculated parameters which can be bewildering for the new user. Moreover, its accessibility can lead to misuse and misinterpretation when the underlying photosynthetic processes are not fully appreciated. This review is timely because it sits at a point of renewed interest in chlorophyll fluorescence where fast measurements of photosynthetic performance are now required for crop improvement purposes. Here we help the researcher make choices in terms of protocols using the equipment and expertise available, especially for field measurements. We start with a basic overview of the principles of fluorescence analysis and provide advice on best practice for taking pulse amplitude-modulated measurements. We also discuss a number of emerging techniques for contemporary crop and ecology research, where we see continual development and application of analytical techniques to meet the new challenges that have arisen in recent years. We end the review by briefly discussing the emerging area of monitoring fluorescence, chlorophyll fluorescence imaging, field phenotyping, and remote sensing of crops for yield and biomass enhancement.
The most obvious event of leaf senescence is the loss of chlorophyll. Chlorophyll degradation proceeds in a well-characterized pathway that, although being common to higher plants, yields a species-specific set of chlorophyll catabolites, termed phyllobilins. Analysis of chlorophyll degradation and phyllobilin accumulation by high-performance liquid chromatography (HPLC) is a valuable tool to investigate senescence processes in plants. In this chapter, methods for the extraction, separation, and quantification of chlorophyll and its degradation products are described. Because of their different physicochemical properties, chlorin-type pigments (chlorophylls and magnesium-free pheo-pigments) and phyllobilins (linear tetrapyrroles) are analyzed separately. Specific spectral properties and polarity differences allow the identification of the different classes of known chlorins and phyllobilins. The methods provided facilitate the analysis of chlorophyll degradation and the identification of chlorophyll catabolites in a wide range of plant species, in different tissues, and under a variety of physiological conditions that involve loss of chlorophyll.
This paper describes the proof-of-concept performance of a low-cost phase fluorometer designed to capture the fluorescence lifetime of chlorophyll in various stages of healthy marine life. The proof-of-concept experimental demonstration is completed using fluoroscein as a close simulant of chlorophyll. Results are extrapolated analytically using simulation to project performance limits (detection and lifetime) in chlorophyll rich environments. The designed system is able to compete with the fundamental performance limits of existing fluorometers designed for chlorophyll analysis while reducing power consumption by a factor of 20, power supply by an order of magnitude or more (to 12 V), and cost by a factor of ten (to a target low-volume system cost of 1000).
Chlorophyll a fluorescence (ChlF) has been used for decades to study the organization, functioning, and physiology of photosynthesis at the leaf and subcellular levels. ChlF is now measurable from remote sensing platforms. This provides a new optical means to track photosynthesis and gross primary productivity of terrestrial ecosystems. Importantly, the spatiotemporal and methodological context of the new applications is dramatically different compared with most of the available ChlF literature, which raises a number of important considerations. Although we have a good mechanistic understanding of the processes that control the ChlF signal over the short term, the seasonal link between ChlF and photosynthesis remains obscure. Additionally, while the current understanding of in vivo ChlF is based on pulse amplitude-modulated (PAM) measurements, remote sensing applications are based on the measurement of the passive solar-induced chlorophyll fluorescence (SIF), which entails important differences and new challenges that remain to be solved. In this review we introduce and revisit the physical, physiological, and methodological factors that control the leaf-level ChlF signal in the context of the new remote sensing applications. Specifically, we present the basis of photosynthetic acclimation and its optical signals, we introduce the physical and physiological basis of ChlF from the molecular to the leaf level and beyond, and we introduce and compare PAM and SIF methodology. Finally, we evaluate and identify the challenges that still remain to be answered in order to consolidate our mechanistic understanding of the remotely sensed SIF signal.
Silver nanoparticles (Ag NPs) are among the most widely produced and used nanomaterial due to their antimicrobial and antibacterial properties, allowing a wide range of commercial applications. Thereby, the increasing use of Ag NPs should inevitably lead to the release and accumulation of these NPs into the environment, resulting in adverse effects on plants, animals and humans. Chlorophyll fluorescence (ChlF) has been proposed as a non-destructive and accurate tool for detecting the impacts of environmental stress on plants. Little is known about the photophysical behavior of plants when exposed to a metallic NPs-containing environment. The present study evaluated the interaction between chlorophyll (Chl) and Ag NPs, over a wide range of nanoparticle concentrations (from 0 μM to 200.0 μM), by monitoring the ChlF. The results reveal that the ChlF is quenched in the presence of Ag NPs, as a result of the static and dynamic quenching processes. The present results suggest that ChlF has a great potential to be used in the future as an analytical tool for monitoring the interaction of plants and NPs as well as investigating the effects of NPs on plants.
Photosynthesis simulations by terrestrial biosphere models are usually based on the Farquhar's model, in which the maximum rate of carboxylation (Vcmax) is a key control parameter of photosynthetic capacity. Even though Vcmax is known to vary substantially in space and time in response to environmental controls, it is typically parameterized in models with tabulated values associated to plant functional types. Remote sensing can be used to produce a spatially continuous and temporally resolved view on photosynthetic efficiency, but traditional vegetation observations based on spectral reflectance lack a direct link to plant photochemical processes. Alternatively, recent space‐borne measurements of sun‐induced chlorophyll fluorescence (SIF) can offer an observational constraint on photosynthesis simulations. Here, we show that top‐of‐canopy SIF measurements from space are sensitive to Vcmax at the ecosystem level, and present an approach to invert Vcmax from SIF data. We use the Soil‐Canopy Observation of Photosynthesis and Energy (SCOPE) balance model to derive empirical relationships between seasonal Vcmax and SIF which are used to solve the inverse problem. We evaluate our Vcmax estimation method at six agricultural flux tower sites in the midwestern US using spaced‐based SIF retrievals. Our Vcmax estimates agree well with literature values for corn and soybean plants (average values of 37 and 101 μmol m−2 s−1, respectively) and show plausible seasonal patterns. The effect of the updated seasonally varying Vcmax parameterization on simulated gross primary productivity (GPP) is tested by comparing to simulations with fixed Vcmax values. Validation against flux tower observations demonstrate that simulations of GPP and light use efficiency improve significantly when our time‐resolved Vcmax estimates from SIF are used, with R2 for GPP comparisons increasing from 0.85 to 0.93, and for light use efficiency from 0.44 to 0.83. Our results support the use of space‐based SIF data as a proxy for photosynthetic capacity and suggest the potential for global, time‐resolved estimates of Vcmax.
Quantifying gross primary production (GPP) remains a major challenge in global carbon cycle research. Spaceborne monitoring of solar-induced chlorophyll fluorescence (SIF), an integrative photosynthetic signal of molecular origin, can assist in terrestrial GPP monitoring. However, the extent to which SIF tracks spatiotemporal variations in GPP remains unresolved. Orbiting Carbon Observatory-2 (OCO-2)'s SIF data acquisition and fine spatial resolution permit direct validation against ground and airborne observations. Empirical orthogonal function analysis shows consistent spatiotemporal correspondence between OCO-2 SIF and GPP globally. A linear SIF-GPP relationship is also obtained at eddy-flux sites covering diverse biomes, setting the stage for future investigations of the robustness of such a relationship across more biomes. Our findings support the central importance of high-quality satellite SIF for studying terrestrial carbon cycle dynamics.
In situ optical meters are widely used to estimate leaf chlorophyll concentration, but non‐uniform chlorophyll distribution causes optical measurements to vary widely among species for the same chlorophyll concentration. Over 30 studies have sought to quantify the in situ/in vitro (optical/absolute) relationship, but neither chlorophyll extraction nor measurement techniques for in vitro analysis have been consistent among studies. Here we: (1) review standard procedures for measurement of chlorophyll; (2) estimate the error associated with non‐standard procedures; and (3) implement the most accurate methods to provide equations for conversion of optical to absolute chlorophyll for 22 species grown in multiple environments. Tests of five Minolta (model SPAD‐502) and 25 Opti‐Sciences (model CCM‐200) meters, manufactured from 1992 to 2013, indicate that differences among replicate models are less than 5%. We thus developed equations for converting between units from these meter types. There was no significant effect of environment on the optical/absolute chlorophyll relationship. We derive the theoretical relationship between optical transmission ratios and absolute chlorophyll concentration and show how non‐uniform distribution among species causes a variable, non‐linear response. These results link in situ optical measurements with in vitro chlorophyll concentration and provide insight to strategies for radiation capture among diverse species. In situ optical meters are widely used to estimate leaf chlorophyll concentration, but non‐uniform chlorophyll distribution causes a highly non‐linear optical/absolute relationship. Over 30 publications have reported absolute/optical relationships, but in vitro chlorophyll extraction and measurement techniques are not standardized, environmental effects on single‐leaf chlorophyll distribution are not well characterized, and differences among replicate meters have not been rigorously assessed. The results of this study more rigorously link in situ optical measurements with absolute chlorophyll concentration and provide insight to strategies for single‐leaf radiation capture among diverse species.
The blue part of the light spectrum has been associated with leaf characteristics which also develop under high irradiances. In this study blue light dose–response curves were made for the photosynthetic properties and related developmental characteristics of cucumber leaves that were grown at an equal irradiance under seven different combinations of red and blue light provided by light-emitting diodes. Only the leaves developed under red light alone (0% blue) displayed dysfunctional photosynthetic operation, characterized by a suboptimal and heterogeneously distributed dark-adapted F v /F m , a stomatal conductance unresponsive to irradiance, and a relatively low light-limited quantum yield for CO 2 fixation. Only 7% blue light was sufficient to prevent any overt dysfunctional photosynthesis, which can be considered a qualitatively blue light effect. The photosynthetic capacity (A max ) was twice as high for leaves grown at 7% blue compared with 0% blue, and continued to increase with increasing blue percentage during growth measured up to 50% blue. At 100% blue, A max was lower but photosynthetic functioning was normal. The increase in A max with blue percentage (0–50%) was associated with an increase in leaf mass per unit leaf area (LMA), nitrogen (N) content per area, chlorophyll (Chl) content per area, and stomatal conductance. Above 15% blue, the parameters A max , LMA, Chl content, photosynthetic N use efficiency, and the Chl:N ratio had a comparable relationship as reported for leaf responses to irradiance intensity. It is concluded that blue light during growth is qualitatively required for normal photosynthetic functioning and quantitatively mediates leaf responses resembling those to irradiance intensity.
It is unclear to what extent seasonal water stress impacts on plant productivity over Amazonia. Using new Greenhouse gases Observing SATellite (GOSAT) satellite measurements of sun-induced chlorophyll fluorescence, we show that midday fluorescence varies with water availability, both of which decrease in the dry season over Amazonian regions with substantial dry season length, suggesting a parallel decrease in gross primary production (GPP). Using additional SeaWinds Scatterometer onboard QuikSCAT satellite measurements of canopy water content, we found a concomitant decrease in daily storage of canopy water content within branches and leaves during the dry season, supporting our conclusion. A large part (r(2) = 0.75) of the variance in observed monthly midday fluorescence from GOSAT is explained by water stress over moderately stressed evergreen forests over Amazonia, which is reproduced by model simulations that include a full physiological representation of photosynthesis and fluorescence. The strong relationship between GOSAT and model fluorescence (r(2) = 0.79) was obtained using a fixed leaf area index, indicating that GPP changes are more related to environmental conditions than chlorophyll contents. When the dry season extended to drought in 2010 over Amazonia, midday basin-wide GPP was reduced by 15 per cent compared with 2009.