Characterization of the Flash-Induced Fluorescence Wave Phenomenon in the Coral Endosymbiont Algae, Symbiodiniaceae.

The dinoflagellate algae, Symbiodiniaceae, are significant symbiotic partners of corals due to their photosynthetic capacity. The photosynthetic processes of the microalgae consist of linear electron transport, which provides the energetic balance of ATP and NADPH production for CO2 fixation, and alternative electron transport pathways, including cyclic electron flow, which ensures the elevated ATP requirements under stress conditions. Flash-induced chlorophyll fluorescence relaxation is a non-invasive tool to assess the various electron transport pathways. A special case of fluorescence relaxation, the so-called wave phenomenon, was found to be associated with the activity of NAD(P)H dehydrogenase (NDH) in microalgae. We showed previously that the wave phenomenon existed in Symbiodiniaceae under acute heat stress and microaerobic conditions, however, the electron transport processes related to the wave phenomenon remained unknown. In this work, using various inhibitors, we show that (i) the linear electron transport has a crucial role in the formation of the wave, (ii) the inhibition of the donor side of Photosystem II did not induce the wave, whereas inhibition of the Calvin-Benson cycle accelerated it, (iii) the wave phenomenon was related to the operation of type II NDH (NDH-2). We therefore propose that the wave phenomenon is an important marker of the regulation of electron transport in Symbiodiniaceae.

Tyr244 of the D2 Protein Is Required for Correct Assembly and Operation of the Quinone-Iron-Bicarbonate Acceptor Complex of Photosystem II.

Two plastoquinone electron acceptors, QA and QB, are present in Photosystem II (PS II) with their binding sites formed by the D2 and D1 proteins, respectively. A hexacoordinate non-heme iron is bound between QA and QB by D2 and D1, each providing two histidine ligands, and a bicarbonate that is stabilized via hydrogen bonds with D2-Tyr244 and D1-Tyr246. Both tyrosines and bicarbonate are conserved in oxygenic photosynthetic organisms but absent from the corresponding quinone-iron electron acceptor complex of anoxygenic photosynthetic bacteria. We investigated the role of D2-Tyr244 by introducing mutations in the cyanobacterium Synechocystis sp. PCC 6803. Alanine, histidine, and phenylalanine substitutions were introduced creating the Y244A, Y244H, and Y244F mutants. Electron transfer between QA and QB was impaired, the back-reaction with the S2 state of the oxygen-evolving complex was modified, and PS II assembly was disrupted, with the Y244A strain being more affected than the Y244F and Y244H mutants. The strains were also highly susceptible to photodamage in the presence of PS II-specific electron acceptors. Thermoluminescence and chlorophyll a fluorescence decay measurements indicated that the redox potential of the QA/QA- couple became more positive in the Y244F and Y244H mutants, consistent with bicarbonate binding being impacted. The replacement of Tyr244 by alanine also led to an insertion of two amino acid repeats from Gln239 to Ala249 within the DE loop of D2, resulting in an inactive PS II complex that lacked PS II-specific variable fluorescence. The 66 bp insertion giving rise to the inserted amino acids therefore created an obligate photoheterotrophic mutant.

Characterization of the Wave Phenomenon in Flash-Induced Fluorescence Relaxation and Its Application to Study Cyclic Electron Pathways in Microalgae.

Photosynthesis is a series of redox reactions, in which several electron transport processes operate to provide the energetic balance of light harvesting. In addition to linear electron flow, which ensures the basic functions of photosynthetic productivity and carbon fixation, alternative electron transport pathways operate, such as the cyclic electron flow (CEF), which play a role in the fine tuning of photosynthesis and balancing the ATP/NADPH ratio under stress conditions. In this work, we characterized the electron transport processes in microalgae species that have high relevance in applied research and industry (e.g., Chlorella sorokiniana, Haematococcus pluvialis, Dunaliella salina, Nannochloropsis sp.) by using flash-induced fluorescence relaxation kinetics. We found that a wave phenomenon appeared in the fluorescence relaxation profiles of microalgae to different extents; it was remarkable in the red cells of H. pluvialis, D. salina and C. sorokiniana, but it was absent in green cells of H. pluvialis and N. limnetica. Furthermore, in microalgae, unlike in cyanobacteria, the appearance of the wave required the partial decrease in the activity of Photosystem II, because the relatively high Photosystem II/Photosystem I ratio in microalgae prevented the enhanced oxidation of the plastoquinone pool. The wave phenomenon was shown to be related to the antimycin A-sensitive pathway of CEF in C. sorokiniana but not in other species. Therefore, the fluorescence wave phenomenon appears to be a species-specific indicator of the redox reactions of the plastoquinone pool and certain pathways of cyclic electron flow.

Crop breeding for a changing climate in the Pannonian region: towards integration of modern phenotyping tools.

The Pannonian Plain, as the most productive region of Southeast Europe, has a long tradition of agronomic production as well as agronomic research and plant breeding. Many research institutions from the agri-food sector of this region have a significant impact on agriculture. Their well-developed and fruitful breeding programmes resulted in productive crop varieties highly adapted to the specific regional environmental conditions. Rapid climatic changes that occurred during the last decades led to even more investigations of complex interactions between plants and their environments and the creation of climate-smart and resilient crops. Plant phenotyping is an essential part of botanical, biological, agronomic, physiological, biochemical, genetic, and other omics approaches. Phenotyping tools and applied methods differ among these disciplines, but all of them are used to evaluate and measure complex traits related to growth, yield, quality, and adaptation to different environmental stresses (biotic and abiotic). During almost a century-long period of plant breeding in the Pannonian region, plant phenotyping methods have changed, from simple measurements in the field to modern plant phenotyping and high-throughput non-invasive and digital technologies. In this review, we present a short historical background and the most recent developments in the field of plant phenotyping, as well as the results accomplished so far in Croatia, Hungary, and Serbia. Current status and perspectives for further simultaneous regional development and modernization of plant phenotyping are also discussed.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Characterization of the wave phenomenon of flash-induced chlorophyll fluorescence in Chlamydomonas reinhardtii.

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, QA- by QB and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of QA- by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321-6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants.

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium.

Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana.

Singlet oxygen (1O2) is an important damaging agent, which is produced during illumination by the interaction of the triplet excited state pigment molecules with molecular oxygen. In cells of photosynthetic organisms 1O2 is formed primarily in chlorophyll containing complexes, and damages pigments, lipids, proteins and other cellular constituents in their environment. A useful approach to study the physiological role of 1O2 is the utilization of external photosensitizers. In the present study, we employed a multiwell plate-based screening method in combination with chlorophyll fluorescence imaging to characterize the effect of externally produced 1O2 on the photosynthetic activity of isolated thylakoid membranes and intact Chlorella sorokiniana cells. The results show that the external 1O2 produced by the photosensitization reactions of Rose Bengal damages Photosystem II both in isolated thylakoid membranes and in intact cells in a concentration dependent manner indicating that 1O2 plays a significant role in photodamage of Photosystem II.

Identification of the AG afterglow thermoluminescence band in the cyanobacterium Synechocystis PCC 6803.

The so-called afterglow, AG, thermoluminescence (TL) band is a useful indicator of the presence of cyclic electron flow (CEF), which is mediated by the NADH dehydrogenase-like (NDH) complex in higher plants. Although NDH-dependent CEF occurs also in cyanobacteria, the AG band has previously not been found in these organisms. In the present study, we tested various experimental conditions and could identify a TL component with ca. +40°C peak temperature in Synechocystis PCC 6803 cells, which were illuminated by far-red (FR) light at around -10°C. The +40°C band could be observed when WT cells were grown under ambient air level CO2 , but was absent in the M55 mutant, which is deficient in the NDH-1 complex. These experimental observations match the characteristics of the AG band of higher plants. Therefore, we conclude that the newly identified +40°C TL component in Synechocystis PCC 6803 is the cyanobacterial counterpart of the plant AG band and originates from NDH-1-mediated CEF. The cyanobacterial AG band was most efficiently induced when FR illumination was applied at -10°C and its contribution to the total TL intensity declined when cells were illuminated above and below this temperature. Based on this phenomenon we also conclude that CEF is blocked by low temperatures at two different sites in Synechocystis PCC 6803: (1) Below -10°C at the level of NDH-1 and (2) below -30°C at the donor or acceptor side of Photosystem I.

Proline is a quencher of singlet oxygen and superoxide both in in vitro systems and isolated thylakoids.

Proline is a versatile plant metabolite, which is produced in large amounts in plants exposed to osmotic and oxidative stress. Proline has been shown to provide protection against various reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals. On the other hand, its protective effect against singlet oxygen has been debated, and it is considered ineffective against superoxide. Here we used various methods for the detection of singlet oxygen (electron paramagnetic resonance, EPR, spin trapping by 2,2,6,6-tetramethyl-4-piperidone, fluorescence probing by singlet oxygen sensor green, SOSG, and oxygen uptake due to chemical trapping) and superoxide (oxygen uptake due to oxygen reduction) in vitro and in isolated thylakoids. We demonstrated that proline does quench both singlet oxygen and superoxide in vitro. By comparing the effects of chemical scavengers and physical quenchers, we concluded that proline eliminates singlet oxygen via a physical mechanism, with a bimolecular quenching rate of ca. 1.5-4 106 M-1 s-1 . Our data also show that proline can eliminate superoxide in vitro in a process that is likely to proceed via an electron transfer reaction. We could also show that proline does quench both singlet oxygen and superoxide produced in isolated thylakoids. The scavenging efficiency of proline is relatively small on a molar basis, but considering its presence in high amounts in plant cells under stress conditions it may provide a physiologically relevant contribution to ROS scavenging, supplementing other nonenzymatic ROS scavengers of plant cells.

Chloramphenicol enhances Photosystem II photodamage in intact cells of the cyanobacterium Synechocystis PCC 6803.

The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the cyanobacterium Synechocystis PCC 6803. Light-induced loss of PSII activity was compared in the presence of chloramphenicol and another protein synthesis inhibitor, lincomycin, by measuring the rate of oxygen evolution in Synechocystis 6803 cells. Our data show that the rate of PSII photodamage was significantly enhanced by chloramphenicol, at the usually applied 200 µg mL-1 concentration, relative to that obtained in the presence of lincomycin. Chloramphenicol-induced enhancement of photodamage has been observed earlier in isolated PSII membrane particles, and has been assigned to the damaging effect of chloramphenicol-mediated superoxide production (Rehman et al. 2016, Front Plant Sci 7:479). This effect points to the involvement of superoxide as damaging agent in the presence of chloramphenicol also in Synechocystis cells. The chloramphenicol-induced enhancement of photodamage was observed not only in wild-type Synechocystis 6803, which contains both Photosystem I (PSI) and PSII, but also in a PSI-less mutant which contains only PSII. Importantly, the rate of PSII photodamage was also enhanced by the absence of PSI when compared to that in the wild-type strain under all conditions studied here, i.e., without addition and in the presence of protein synthesis inhibitors. We conclude that chloramphenicol enhances photodamage mostly by its interaction with PSII, leading probably to superoxide production. The presence of PSI is also an important regulatory factor of PSII photodamage most likely via decreasing excitation pressure on PSII.

A multi-parametric screening platform for photosynthetic trait characterization of microalgae and cyanobacteria under inorganic carbon limitation.

Microalgae and cyanobacteria are considered as important model organisms to investigate the biology of photosynthesis; moreover, they are valuable sources of biomolecules for several biotechnological applications. Understanding the species-specific traits of photosynthetic electron transport is extremely important, because it contributes to the regulation of ATP/NADPH ratio, which has direct/indirect links to carbon fixation and other metabolic pathways and thus overall growth and biomass production. In the present work, a cuvette-based setup is developed, in which a combination of measurements of dissolved oxygen, pH, chlorophyll fluorescence and NADPH kinetics can be performed without disturbing the physiological status of the sample. The suitability of the system is demonstrated using a model cyanobacterium Synechocystis sp. PCC6803, as well as biofuel-candidate microalgae species, such as Chlorella sorokiniana, Dunaliella salina and Nannochloropsis limnetica undergoing inorganic carbon (Ci) limitation. Inorganic carbon limitation, induced by photosynthetic Ci uptake under continuous illumination, caused a decrease in the effective quantum yield of PSII (Y(II)) and loss of oxygen-evolving capacity in all species investigated here; these effects were largely recovered by the addition of NaHCO3. Detailed analysis of the dark-light and light-dark transitions of NADPH production/uptake and changes in chlorophyll fluorescence kinetics revealed species- and condition-specific responses. These responses indicate that the impact of decreased Calvin-Benson cycle activity on photosynthetic electron transport pathways involving several sections of the electron transport chain (such as electron transfer via the QA-QB-plastoquinone pool, the redox state of the plastoquinone pool) can be analyzed with high sensitivity in a comparative manner. Therefore, the integrated system presented here can be applied for screening for specific traits in several significant species at different stages of inorganic carbon limitation, a condition that strongly impacts primary productivity.

A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.

Stabilization of Photosystem II by the PsbT protein impacts photodamage, repair and biogenesis.

Photosystem II (PS II) catalyzes the light-driven process of water splitting in oxygenic photosynthesis. Four core membrane-spanning proteins, including D1 that binds the majority of the redox-active co-factors, are surrounded by 13 low-molecular-weight (LMW) proteins. We previously observed that deletion of the LMW PsbT protein in the cyanobacterium Synechocystis sp. PCC 6803 slowed electron transfer between the primary and secondary plastoquinone electron acceptors QA and QB and increased the susceptibility of PS II to photodamage. Here we show that photodamaged ∆PsbT cells exhibit unimpaired rates of oxygen evolution if electron transport is supported by HCO3- even though the cells exhibit negligible variable fluorescence. We find that the protein environment in the vicinity of QA and QB is altered upon removal of PsbT resulting in inhibition of QA- oxidation in the presence of 2,5-dimethyl-1,4-benzoquinone, an artificial PS II-specific electron acceptor. Thermoluminescence measurements revealed an increase in charge recombination between the S2 oxidation state of the water-oxidizing complex and QA- by the indirect radiative pathway in ∆PsbT cells and this is accompanied by increased 1O2 production. At the protein level, both D1 removal and replacement, as well as PS II biogenesis, were accelerated in the ∆PsbT strain. Our results demonstrate that PsbT plays a key role in optimizing the electron acceptor complex of the acceptor side of PS II and support the view that repair and biogenesis of PS II share an assembly pathway that incorporates both de novo synthesis and recycling of the assembly modules associated with the core membrane-spanning proteins.

A single plasmid based CRISPR interference in Synechocystis 6803 - A proof of concept.

We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a "proof of concept". Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex.

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, QA, and the secondary plastoquinone acceptor, QB, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced QB binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between QA and QB, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.

Co-occurrence of Mild Salinity and Drought Synergistically Enhances Biomass and Grain Retardation in Wheat.

In the present study we analyzed the responses of wheat to mild salinity and drought with special emphasis on the so far unclarified interaction of these important stress factors by using high-throughput phenotyping approaches. Measurements were performed on 14 genotypes of different geographic origin (Austria, Azerbaijan, and Serbia). The data obtained by non-invasive digital RGB imaging of leaf/shoot area reflect well the differences in total biomass measured at the end of the cultivation period demonstrating that leaf/shoot imaging can be reliably used to predict biomass differences among different cultivars and stress conditions. On the other hand, the leaf/shoot area has only a limited potential to predict grain yield. Comparison of gas exchange parameters with biomass accumulation showed that suppression of CO2 fixation due to stomatal closure is the principal cause behind decreased biomass accumulation under drought, salt and drought plus salt stresses. Correlation between grain yield and dry biomass is tighter when salt- and drought stress occur simultaneously than in the well-watered control, or in the presence of only salinity or drought, showing that natural variation of biomass partitioning to grains is suppressed by severe stress conditions. Comparison of yield data show that higher biomass and grain yield can be expected under salt (and salt plus drought) stress from those cultivars which have high yield parameters when exposed to drought stress alone. However, relative yield tolerance under drought stress is not a good indicator of yield tolerance under salt (and salt plus drought) drought stress. Harvest index of the studied cultivars ranged between 0.38 and 0.57 under well watered conditions and decreased only to a small extent (0.37-0.55) even when total biomass was decreased by 90% under the combined salt plus drought stress. It is concluded that the co-occurrence of mild salinity and drought can induce large biomass and grain yield losses in wheat due to synergistic interaction of these important stress factors. We could also identify wheat cultivars, which show high yield parameters under the combined effects of salinity and drought demonstrating the potential of complex plant phenotyping in breeding for drought and salinity stress tolerance in crop plants.

Differential gene expression and physiological changes during acute or persistent plant virus interactions may contribute to viral symptom differences.

Viruses have different strategies for infecting their hosts. Fast and acute infections result in the development of severe symptoms and may cause the death of the plant. By contrast, in a persistent interaction, the virus can survive within its host for a long time, inducing only mild symptoms. In this study, we investigated the gene expression changes induced in CymRSV-, crTMV-, and TCV-infected Nicotiana benthamiana and in PVX- and TMV-U1-infected Solanum lycopersicum plants after the systemic spread of the virus by two different high-throughput methods: microarray hybridization or RNA sequencing. Using these techniques, we were able to clearly differentiate between acute and persistent infections. We validated the gene expression changes of selected genes by Northern blot hybridization or by qRT-PCR. We show that, in contrast to persistent infections, the drastic shut-off of housekeeping genes, downregulation of photosynthesis-related transcripts and induction of stress genes are specific outcomes with acute infections. We also show that these changes are not a consequence of host necrosis or the presence of a viral silencing suppressor. Thermal imaging data and chlorophyll fluorescence measurements correlated very well with the molecular changes. We believe that the molecular and physiological changes detected during acute infections mostly contribute to virus symptom development. The observed characteristic physiological changes associated with economically more dangerous acute infections could serve as a basis for the elaboration of remote monitoring systems suitable for detecting developing virus infections in crops. Moreover, as molecular and physiological changes are characteristics of different types of virus lifestyles, this knowledge can support risk assessments of recently described novel viruses.

Environmental pH and a Glu364 to Gln mutation in the chlorophyll-binding CP47 protein affect redox-active TyrD and charge recombination in Photosystem II.

In Photosystem II, loop E of the chlorophyll-binding CP47 protein is located near a redox-active tyrosine, YD , forming a symmetrical analog to loop E in CP43, which provides a ligand to the oxygen-evolving complex (OEC). A Glu364 to Gln substitution in CP47, near YD , does not affect growth in the cyanobacterium Synechocystis sp. PCC 6803; however, deletion of the extrinsic protein PsbV in this mutant leads to a strain displaying a pH-sensitive phenotype. Using thermoluminescence, chlorophyll fluorescence, and flash-induced oxygen evolution analyses, we demonstrate that Glu364 influences the stability of YD and the redox state of the OEC, and highlight the effects of external pH on photosynthetic electron transfer in intact cyanobacterial cells.

Maximum fluorescence and electron transport kinetics determined by light-induced fluorescence transients (LIFT) for photosynthesis phenotyping.

Photosynthetic phenotyping requires quick characterization of dynamic traits when measuring large plant numbers in a fluctuating environment. Here, we evaluated the light-induced fluorescence transient (LIFT) method for its capacity to yield rapidly fluorometric parameters from 0.6 m distance. The close approximation of LIFT to conventional chlorophyll fluorescence (ChlF) parameters is shown under controlled conditions in spinach leaves and isolated thylakoids when electron transport was impaired by anoxic conditions or chemical inhibitors. The ChlF rise from minimum fluorescence (Fo) to maximum fluorescence induced by fast repetition rate (Fm-FRR) flashes was dominated by reduction of the primary electron acceptor in photosystem II (QA). The subsequent reoxidation of QA- was quantified using the relaxation of ChlF in 0.65 ms (Fr1) and 120 ms (Fr2) phases. Reoxidation efficiency of QA- (Fr1/Fv, where Fv = Fm-FRR - Fo) decreased when electron transport was impaired, while quantum efficiency of photosystem II (Fv/Fm) showed often no significant effect. ChlF relaxations of the LIFT were similar to an independent other method. Under increasing light intensities, Fr2'/Fq' (where Fr2' and Fq' represent Fr2 and Fv in the light-adapted state, respectively) was hardly affected, whereas the operating efficiency of photosystem II (Fq'/Fm') decreased due to non-photochemical quenching. Fm-FRR was significantly lower than the ChlF maximum induced by multiple turnover (Fm-MT) flashes. However, the resulting Fv/Fm and Fq'/Fm' from both flashes were highly correlated. The LIFT method complements Fv/Fm with information about efficiency of electron transport. Measurements in situ and from a distance facilitate application in high-throughput and automated phenotyping.