The requirements for imaging are identical to those described above for use with any fluorometer. However, below we have outlined additional requirements that need to be followed in order to obtain meaningful results. As imaging is working on a relatively large illuminated surface, it is critical that the leaf or material of interest is held horizontal to the actinic and modulated lights in order to prevent heterogeneous illumination over different areas of the leaf.
The material should also be held at the correct height relative to the light and camera in instruments that rely on in-built light calibrations based on a distance and do not provide a measurement of PAR at the measurement surface. If you are working with C 4 plants, you may need higher saturating pulses.
However, many commercial imaging systems rely on banks or panels of LEDs that provide the measuring beam and the actinic source, and they tend to be on one waveband and are therefore not capable of carrying out an FR pulse. This is important for measurements of primary productivity. If we consider that the measurement of radiation use efficiency RUE of a plant canopy is weight of dry matter per unit radiation absorbed by the canopy, then to understand how cumulative short-term changes in photosynthesis, for example caused by mid-day depression of photosynthesis or canopy development, impact RUE we will need to quantify these processes on a daily basis Black et al.
Limitations of single instruments can be caused by power supply and low numbers of measuring sensors. However, there are now devices available on the market that have been designed for long-term monitoring of fluorescence using the modulated technique described above, for example the Walz Monitoring PAM Porcar-Castell et al. The design is modular, with a series of measuring heads, each containing the optics and light sources required for measurement of key parameters and connected physically to a data collection unit or a computer.
It is possible to power the entire system using solar panels and upload the data remotely, requiring no user interaction except in cases where the leaf becomes dislodged. In practice, several measuring heads are deployed in vegetation and fixed to a leaf where they will remain until the user moves them.
Murchie, unpublished data. This is promising for some applications and offers the unique ability to assess photosynthesis continuously over large areas, although it clearly has severe limitations in comparison with the accuracy and precision of leaf-level measurements. Nevertheless, correlation of key drought-related parameters with chlorophyll fluorescence signal could be obtained using a UAV Zarco-Tejada et al.
Such passive techniques have the disadvantage of uneven illumination of the field of view and no saturating pulse. Laser-induced fluorescence transient LIFT is a technique that uses a laser to project an excitation beam e. Fluorescence emission is collected using a telescope, filtered, and detected using a large photodiode. Data are processed in real time for an immediate, remote measurement of PSII yield and electron transport.
This technique compares well with that made by routine fluorometry, and so far it has been used successfully for ecophysiological studies and remote sensing from distances of 5—50 m or more Ananyev et al. This is a bespoke system, but LIFT hardware, protocols for fluorescence induction, fluorescence data analysis, sensing, and data processing are described in the literature e.
Ananyev et al. This article provides an update on previous reviews on the technique of chlorophyll fluorescence. Inspired by the new applications of this technique in crop phenotyping and monitoring, we have covered the aspects of methodology the user should be aware of, along with some new and emerging variations. This technique lends itself remarkably well to the requirements of crop improvement: the rapid assessment with high throughput of measurements which produce data-rich results.
In particular, we expect that the development of monitoring and remote fluorescence techniques should play a key part in the development of automated crop phenotyping techniques; so far this has yet to be seen. These are included to provide the user with some guidance for good practice and help to overcome some pitfalls in common situations. See Table 1 for a list of formulae.
Usually devices will make calculations automatically, and the diligent user should compare these with the procedure below and refer to Fig.
Pask et al. Check for quenching induced by the measuring beam and adjust the measuring beam intensity accordingly. Attain F m value. If using ambient light, ensure that the light intensity at the leaf surface is known and that it is stable during the measurement, in order that results have proper context.
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Viridis mutants in barley: genetic, fluoroscopic and ultrastructural characterisation. Somerville CR Analysis of photosynthesis with mutants of higher plants and algae. Annu Rev Plant Physiol — Elsevier Biomedical Press, New York, pp — Taylor WC Regulatory interactions between nuclear and plastid genomes.
Dev Genet 8: — Biochemistry — Vermaas W Molecular-biological approaches to analyze photosystem II structure and function. The psbB operon from spinach. Plant Mol Biol 4: — Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Peter Westhoff. The screen was based on the fact that severe perturbations in photosynthetic electron transport, such as those caused by incubating algal cells in the presence of the photosynthetic electron transport inhibitor 3- 3,4-dichlorophenyl -1,1-dimethylurea DCMU , lead to high steady-state levels of Chl fluorescence.
Following its application for screening of Chlamydomonas mutant libraries see for example Harris, the concept was tested in higher plants Miles and Daniel, and employed for the screening of maize Miles and Daniel, ; Barkan et al.
TABLE 1. Chronology of Chl fluorescence phenotyping based gene discovery studies. Even though such hcf mutant screens can be performed rapidly and efficiently, and have significantly enhanced our knowledge of the molecular repertoire required for photosynthesis and chloroplast biogenesis, only mutants with severe defects can be unequivocally detected, and these are often lethal under photoautotrophic conditions.
However, technological progress in Chl fluorescence analyses during the s and s allowed the technique to be employed for more elaborate modes of screening, and led to the identification of algal or plant mutants with relatively modest alterations in photosynthetic performance. For example, Varotto et al. This set-up facilitated the screening of large En transposon or T-DNA mutagenized Arabidopsis populations, and pam mutants disrupted in the nucleus-encoded photosystem I subunits PsaE1 pam4 Varotto et al.
Due to their sessile lifestyle, many multicellular photosynthetic organisms have evolved various strategies to cope with light stress Ort, When the photosynthetic machinery is exposed to excessively high levels of light, short- and long-term adaptive responses are triggered at the molecular level, which allow for the thermal dissipation of excited energy by NPQ mechanisms to prevent over-reduction of the electron transport chain. At least four processes contribute to NPQ: qE, qZ zeaxanthin-dependent quenching , qT state-transition-dependent quenching and qI reviewed in: Ruban, Mutant identification was essentially based on the comparison of two video images of Chl fluorescence captured under different illumination conditions.
The first picture was taken in the dark-adapted state during a saturating light pulse F m , or shortly after the onset of high-light treatment F. Subsequent analyses revealed three distinct groups of mutants with aberrant NPQ reviewed in: Golan et al. One such mutant, pgr1 , was further characterized, and shown to be defective in the photosynthetic electron transfer C PETC gene, which encodes the Rieske subunit of the cytochrome b 6 f complex Munekage et al.
Another mutant line impaired in the build-up of the proton gradient is pgr5 Shikanai et al. The second group with aberrant NPQ comprised the mutants npq1 and npq2 , which display defects in the xanthophyll cycle and are disrupted in the violaxanthin de-epoxidase and zeaxanthin epoxidase, respectively Niyogi et al.
The third type of mutant npq4 showed normal pigment composition, xanthophyll cycle activity and photosynthetic electron transport, but this mutant was nevertheless specifically affected at the level of qE Li et al.
In a subsequent study, which was designed to isolate Arabidopsis lines affected in other slowly reversible NPQ components, CFVI was used to screen mutagenized seedlings for suppressors of the npq4 phenotype Brooks et al.
Several studies Fleischmann et al. Mutants affected in state transitions were identified by comparing fluorescence images taken under state-1 and state-2 conditions, and this type of differential fluorescence screen enabled Fleischmann et al.
These mutants were characterized by high Chl fluorescence levels at room temperature even under state-2 conditions, indicating that they were physiologically locked in state 1. One of these pathways is mediated by the NADH-like dehydrogenase NDH complex, which is also responsible for chlororespiration in the dark. Under such conditions, the fluorescence signal is almost proportional to the reduction state of the plastoquinone pool Krause and Weis, , so that the chlororespiratory activity of the NDH complex can be derived from the degree to which the PIF is depressed in mutants with a dysfunctional NDH complex Shikanai et al.
Screening of over 50, M2 seedlings for aberrant PIFs led to the identification of 17 crr mutants in Arabidopsis. These could be assigned to at least 11 loci, and further analyses revealed the existence of novel NDH subunits and allowed the functional characterization of factors required for efficient NDH complex biogenesis reviewed in: Peng et al.
Plants and algae can undergo photosynthetic acclimation processes which take place over periods of hours or days and entail substantial changes in plastid and nuclear gene expression, as well as adjustments of the photosynthetic apparatus. For instance, the long-term response to high light levels has been thoroughly studied and, instead of reducing the demands on light harvesting, it actually enhances the capacity for electron transport and carbon dioxide fixation.
To investigate the molecular mechanisms behind the signal cascades that activate the acclimation response to high light, Walters et al. Subsequent studies using Arabidopsis double and triple mutants altered in the day and night modes of photoassimilate export from the chloroplast provided evidence that carbohydrates act as chloroplast-to-nucleus retrograde signals and modulate the acclimation response to high light Schmitz et al.
The phosphoglycolate generated by the latter reaction must be degraded via a complex mechanism which is known as photorespiration, because CO 2 is released during the process. The photorespiratory pathway is distributed between four compartments chloroplasts, cytosol, peroxisomes and mitochondria and requires the action of several transporters and enzymes.
Recently, it was shown that mutations in components involved in the photorespiratory pathway also impair photosynthetic light reactions, as revealed by the observation that photorespiratory mutants transferred from high to ambient CO 2 concentrations showed a decline in PSII functionality Takahashi et al. Thus, Badger et al. To this end, levels of PSII function in mutagenized Arabidopsis seedlings grown under high concentrations of CO 2 , and in its absence, were compared.
Two major mutant phenotype classes could be distinguished. As aquatic organisms, many unicellular green algae are characterized by a remarkably flexible metabolism, and can acclimate rapidly to anaerobic conditions Terashima et al. As part of an extensive response to anaerobiosis, expression and synthesis of oxygen-labile [Fe—Fe] hydrogenases are induced in C. Several factors required for expression, maturation and activity of [Fe—Fe] hydrogenases have been identified, most of them through a H 2 -sensing, chemochromic screening system that can discriminate Chlamydomonas mutants with aberrant H 2 production capacities reviewed in: Hemschemeier et al.
An alternative, less time-consuming approach has been demonstrated by Godaux et al. As a proof of concept, screening of a small Chlamydomonas population of about strains generated by insertional mutagenesis yielded five mutants with a Chl fluorescence signature similar to that of the [Fe—Fe] hydrogenase-deficient control strain, and one of them turned out to be defective in the previously characterized [Fe—Fe] hydrogenase assembly factor G HydG Posewitz et al.
Moreover, in various mutants affected in anaerobic energy metabolism, the effective quantum yield of PSII was shown to be correlated with the level of [Fe—Fe] hydrogenase activity. Thus, the screening system represents a time-saving, alternative approach to the chemochromic method, and is capable of detecting mutants impaired in [Fe—Fe] hydrogenase biogenesis, regulation or activity.
Although respiration and photosynthesis take place in different organelles in photosynthetic eukaryotes, the energy metabolisms of mitochondria and chloroplasts are intertwined at multiple levels. Not only do these organelles share over dual-targeted proteins reviewed in: Carrie and Small, , provide both ATP and contribute to photorespiration, chloroplasts can shuttle reducing power to mitochondria via the malate valve reviewed in: Scheibe, ; Kramer and Evans, For instance, in Chlamydomonas mutants defective in different complexes of the respiratory electron transport chain, the resulting ATP deficiency is counterbalanced by increased non-photochemical reduction of the plastoquinone pool mediated by the chlororespiratory pathway, LHCII protein association to PSI and cyclic photophosphorylation Cardol et al.
These results are consistent with the finding that overall fitness and yields of photosynthesis were only significantly reduced when state transitions and mitochondrial respiration were concomitantly impaired in the Chlamydomonas double mutant stt dum22 Cardol et al.
Thus, increased cyclic electron transport rates induced by state 2 transitions can supply extra ATP when respiratory ATP production becomes limiting and, conversely, mitochondrial cooperation is increased when CEF is downregulated in Chlamydomonas. One important conclusion that could be drawn from these studies was that PSII efficiencies were reduced in respiratory mutants and was explained by enhanced rates of non-photochemical reduction of plastoquinone mediated by the chlororespiratory pathway and preferential association of LHCII proteins with PSI Cardol et al.
Massoz et al. Several mutants disrupted in subunits of the respiratory complex I or the isocitrate lyase were isolated from a collection of about insertional mutants generated in either a wild-type or a state transition-defective strain stt A later refinement of the screening procedure used the CEF mutant pgrl1 as the starting strain with a view to isolating mutants impaired in mitochondrial complex I Massoz et al.
Subsequent screening of about insertional mutants created in the pgrl1 background resulted in 46 mutants with reduced PSII efficiency, of which three were complex I mutants. Forward genetic approaches still dominated mutant searches in the late s and early s Lloyd and Meinke, , but thanks to advances in genome sequencing technologies, the establishment of large mutant libraries and the development of new genetic tools such as RNA silencing techniques Mohr et al.
Relative to classical forward genetic approaches, reverse genetic screens start with a significantly reduced number of lines or strains, which are generally disrupted in genes with poorly characterized or unknown functions. Depending on the stringency of preselection criteria e.
In addition, Arabidopsis genes of unknown function were considered together with homologs found in cyanobacteria but not in green algae, since C. Insertion lines were identified for 21 of the 36 genes pre-selected by means of the bioinformatics screen, and these were tested for NDH activity. Since Chl fluorescence-based phenotyping is no longer as time-consuming as it once was, and manageable numbers of candidates can be examined in reverse genetics projects, contemporary screening approaches can be extended to more elaborate protocols in which subtle or multiple photosynthetic phenotypes can be detected in a single, albeit longer, experimental run.
Commercial Chl video imaging systems now make it possible to set up automated measuring routines composed of several analytical blocks that can last for days.
One example of such a combined screening protocol is shown in Figure 1 , which we use routinely for initial phenotyping of selected Arabidopsis mutant lines. Example of a combined screening protocol based on Chl fluorescence video imaging which is able to identify hcf, crr, npq, pam and PSII repair mutants. Plants were dark-adapted for 20 min and acclimated to measuring light for 5 min prior to the analysis. For further explanations, see the main text. B Example of an hcf mutant phenotype, which can be detected in measurement block 1.
C Identification of a crr mutant phenotype in block 2. D Detection of a high NPQ phenotype in block 3. E Example of a pam mutant phenotype, which can be distinguished at the end of block 3.
F Detection of an npq phenotype in measurement block 5. Chl fluorescence signals were normalized to F m and are shown in gray on a scale from 0 to 1 in D—F. In principle, the approach comprises six phases, in which most of the previously described Chl fluorescence signatures of photosynthetic mutants can be identified Figure 1A.
The second analytical block was designed to identify mutant lines with a crr phenotype, and detects NDH activity by means of a PIF measurement Figure 1C. Block 3 implements a standard slow induction experiment, which is carried out under moderate actinic light intensities. Several informative parameters can be extracted in block 3 which reveal aspects of the transient dynamics of photosynthesis upon a dark-light shift. For instance, pgr mutants can be already identified at this stage by their low transient NPQ phenotype DalCorso et al.
The increased NPQ in such mutants can be attributed to a high operating qE, which is established as a result of proton accumulation in the thylakoid lumen already under moderate light intensities. Overall, the CFVI protocol outlined above can already uncover a wide range of phenotypes, but can be further expanded to cover a larger collection of photosynthetic parameters, such as the determination of P max from light saturation curves van Rooijen et al.
The photosynthetic lifestyle of plants and algae requires a high degree of flexibility and the ability to adapt to rapidly fluctuating environments.
However, for reasons of scalability and reproducibility most of the screening studies referred to here were conducted with small Arabidopsis plants or algae grown in stable, standardized laboratory settings and would have been impossible with fully developed crops under field conditions.
Furthermore, phenotyping of mutant collections involved measurements of only one or a few photosynthetic parameters, which were determined at one or more time points, thus providing a rather static picture of the highly dynamic process of photosynthesis. It is therefore obvious that many factors that contribute to the fine tuning of photosynthesis in response to dynamic environmental changes will have not been identified by previous screening procedures Cruz et al.
One logical and straightforward way to bypass this limitation would be to carry out phenotyping of mutant collections in the field, and suitable large-scale Chl fluorescence image analyzers are now available for this task e. However, besides the fact that in several countries the cultivation of genetically modified plants in the field is either prohibited or subject to legal restrictions, such studies are complicated by a multitude of overlapping, unpredictable abiotic and biotic stress factors, and statistical evaluation of the results become particularly challenging.
For these reasons, the dynamic environmental photosynthesis imager DEPI platform was developed for replication of natural, fluctuating growth conditions in the laboratory Cruz et al. Several parameters can be controlled light intensity, CO 2 concentration, humidity and temperature in the growth chamber, and rapid responses as well as long-term acclimation processes of photosynthesis can be assessed in situ by the integrated CFVI system in more than two hundred plants simultaneously.
As a proof of concept, a library of over T-DNA Arabidopsis lines disrupted in nuclear genes coding for chloroplast-targeted proteins Ajjawi et al. PSB33 is a green-lineage-specific protein Merchant et al.
Thus, the DEPI system can reveal new, complex and previously unseen phenotypes, and provides a versatile experimental platform with which to identify factors required for remodeling and regulation of photosynthesis under dynamic environmental conditions. Forward and reverse genetics are efficient strategies for elucidating the functions of a single gene or of small gene families, but these approaches reach their limits when the genetic architecture of a quantitative trait and its interaction with the environment needs to be determined.
Most agronomically important traits e. Natural variation also exists for photosynthetic traits and can be roughly divided into morphological and physiological variations, which have been investigated in several studies with different plant species reviewed in: Flood et al. For instance, Jung and Niyogi examined natural NPQ variation in different Arabidopsis accessions and provided evidence that thermal dissipation is a quantitative trait that depends on multiple, nucleus-encoded genetic factors.
Remarkably, HQE1 and HQE2 were not mapped to previously characterized factors identified in forward genetic approaches, indicating that quantitative genetics can serve as a complementary strategy to dissect the genetic architecture of thermal dissipation. Even though quantitative genetic approaches have a long history in plant science, their potential for photosynthesis research has not yet been fully explored. This may simply reflect the high complexity of the genetic architecture of photosynthesis, which not only comprises the several hundred genes directly involved in biogenesis processes, regulation or acclimation of photosynthesis, but also involves two quite distinct genetic systems plastid and nuclear genome with different inheritance modes.
Moreover, successful quantitative genetic approaches in photosynthesis research require reproducible, non-invasive, high-throughput phenotyping pipelines that were not available until recently.
However, several platforms have been developed in recent years. Examples include FluorImager Barbagallo et al. As an example for the combination of a GWAS and a high-throughput Chl fluorescence phenotyping approach, van Rooijen et al. Of 63 newly identified gene candidates, 13 encode chloroplast-localized proteins, most of which are either associated with abiotic stress responses or have unknown functions. Modern phenotyping systems are highly flexible and will allow the identification of new genotype-phenotype-environment relationships and accelerate gene discovery studies significantly.
Consequently, with the exception of screening approaches under highly specialized conditions, tedious forward genetic screening procedures, which were carried out by single researchers or research groups in the past, will become obsolete. A major task in the future will lie in the processing, handling, quality control, maintenance, storage, analysis and sharing of the vast amount of data collected by CFVI-based phenotyping studies, which will become even more challenging when such screens are combined with other non-invasive phenotyping technologies Walter et al.
But computational techniques for assessing the quality of phenotypic data Xu et al. Chl fluorescence video imaging also has the potential to be an important technological driver in crop science, since it offers an efficient screening technology for rapid evaluation of plant performance under stress conditions such as drought, salinity, freezing, chilling, high temperature or nutrient deficiency reviewed in: Baker and Rosenqvist, CFVI is of particular interest in plant breeding programs, since low-cost and precise high-throughput phenotyping technologies have been regarded as one of the major bottleneck in the postgenomic era of plant breeding reviewed in: Araus and Cairns, A further challenge is the difficulty to extrapolate results gained under a strictly controlled environment such as a growth chamber or greenhouse to field conditions.
It is therefore inevitable to establish high throughput phenotyping technologies under heterogeneous field conditions to analyze quantitative traits and to elucidate their underlying genetic architecture for future breeding efforts. Significant progress in non-invasive sensor and imaging technology has been made reviewed in: White et al. While recent work has mainly focused on scaling up CFVI screening systems for simultaneous evaluation of large sample collections, a future direction might be to explore the potential of screening single cells by exploiting their Chl fluorescence fingerprints.
Flow cytometry technologies are well established for unicellular microalgae in environmental and toxicological studies reviewed in: Hyka et al.
Flow cytometry is generally coupled to fluorescence-activated cell sorting, which permits the isolation of a desired cell population with specific physiological properties. Recently, this technique has been successfully employed to screen high-lipid Chlamydomonas mutants that were stained with the lipid-sensitive dye Nile Red prior to screening Xie et al.
Moreover, Chl autofluorescence has been used in flow cytometry studies as an endogenous probe to sort tobacco mesophyll protoplasts Harkins et al. Although implementation will be challenging, the combination of flow cytometry and Chl fluorescence kinetics-based cell sorting can provide a fast means of screening mutagenized cell populations for specific Chl fluorescence phenotypes.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ajjawi, I. Large-scale reverse genetics in Arabidopsis: case studies from the Chloroplast Project. Plant Physiol. Allen, J. Protein phosphorylation in regulation of photosynthesis. Acta , — Araus, J.
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Baker, N. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. Barbagallo, R. Rapid noninvasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging. Barkan, A.
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