Analyzing photosynthetic performance in natural fluctuating environment using light-induced fluorescence transient (LIFT) method in high-throughput

In the present work, the knowledge gap concerning the interaction of photosynthesis with its fluctuating environment was filled by acquiring over one million chlorophyll fluorescence measurements in semi-field and field conditions. Five crop species were monitored in high spatio-temporal resolution. Hereby, the light-induced fluorescence transient (LIFT) method was established as high-throughput system scanning over the crop canopy. The LIFT method uses a series of excitation flashlets to induce variable fluorescence (Fv) and to monitor fluorescence relaxation (Fr). The resulting fluorescence transient reflects the coupled kinetics of primary quinone electron acceptor (QA) reduction and its subsequent reoxidation. Fv normalized with the induced maximum fluorescence level results in the quantum efficiency of the photosystem II (Fv/Fm in the dark and Fq'/Fm' in the light) reflecting the amount of photosynthetically transported electrons per photon. The local fluorescence maximum (FmQA) and maximum fluorescence (Fm) were induced from 60 cm distance using LIFT flashes differing in excitation length and power. FmQA did not fully reduce the electron transport chain which enabled the determination of the reoxidation efficiency 5 ms after QA reduction (Fr2/Fm in the dark respective Fr2'/Fm' in the light). This newly established parameter was dependent on the functionality of the electron transport chain and temperature. In contrast, Fq'/Fm' was mainly dependent on light intensity. Under controlled conditions, electron transport rates (ETR) based on Fq'/Fm' correlated to ETR retrieved from CO2 assimilation measurements. For the first time, a sufficiently large data set including spectral measurements was collected under semi-field conditions to identify factors determining the diurnal and seasonal photosynthesis pattern. According to Lasso regression analysis, Fq'/Fm' was dependent on photosynthetic photon flux density (PPFD) and spectral indices. The designed linear model accounted for almost 50% of the variance in Fq'/Fm' measured over two growing seasons. The second parameter, Fr2/Fm respective Fr2'/Fm', was highly determined by temperature and crop species, e.g. separating the response of winter hard rapeseed and soybean at lower temperatures. Only minor influence on the measured parameters was detected for different years, daytime, measuring date and hence seasonal or plant development stage. In the following, genotypic differences were detected on the parameter mean or the interaction of the parameters with environmental factors. Especially in soybean, genotypic differences in Fq'/Fm' and Fr2'/Fm' were more consistently detected when instead of the mean, the interaction with PPFD and temperature was considered. Analyzing the mean of selected time periods was useful for detection of stress response. Increasing drought stress decreased Fq'/Fm' under controlled and semi-field conditions in maize. In contrast, Fr2/Fm respective Fr2'/Fm' increased in response to drought probably reflecting enhanced cyclic electron transport. Powdery mildew infection was detected by Fq'/Fm' before symptoms were visible by eye. Drought response of photosynthesis was also detected in soybean under field conditions. Summarizing all collected field data, Fq'/Fm' was still dependent on PPFD, but even stronger correlated to reflectance of sunlight at 685 nm on the target leaf. The response of Fr2/Fm respective Fr2'/Fm' to temperature persisted, and explained 79% of all variance in maize. The LIFT screening approach identified tolerant genotypes regarding light and temperature use efficiency under control and stress conditions. Analyzing the response curves of the considered parameters related to PPFD and temperature allows the prediction of photosynthetic performance and optimization of genotypic selection in various environments.In der vorliegenden Arbeit wurde die Wissenslücke bezüglich der Photosynthese Wechselwirkung mit ihrer fluktuierenden Umgebung durch die Erfassung von über einer Million Chlorophyllfluoreszenzmessungen unter Feldähnlichen- und Feldbedingungen bearbeitet. Fünf Nutzpflanzenarten wurden in einer hohen raumzeitlichen Auflösung beobachtet. Dabei wurde die lichtinduzierte Fluoreszenztransienten (LIFT) Methode als ein Hochdurchsatz-System etabliert, das über den Pflanzenbestand scannt. Die LIFT-Methode verwendet eine Reihe von Anregungslichtblitzen, um eine variable Fluoreszenz (Fv) zu induzieren. Anschließend wird die Fluoreszenzrelaxation (Fr) beobachtet. Der resultierende Fluoreszenztransient spiegelt die gekoppelte Kinetik der Reduktion des primären Chinon-Elektronenakzeptors (QA) und die anschließende Reoxidation wider. Fv normalisiert mit dem induzierten Fluoreszenzmaximum ergibt die Quanteneffizienz des Photosystems II (Fv/Fm im Dunkeln und Fq'/Fm' im Licht), was die Menge der photosynthetisch transportierten Elektronen pro Photon widerspiegelt. Das lokale Fluoreszenzmaximum (FmQA) und die maximale Fluoreszenz (Fm) wurden aus 60 cm Entfernung unter Verwendung von LIFT-Lichtblitzen induziert, die sich in Anregungslänge und Leistung unterschieden. FmQA reduzierte die Elektronentransportkette nicht vollständig, was die Bestimmung der Reoxidationseffizienz 5 ms nach der QA-Reduktion (Fr2/Fm im Dunkeln bzw. Fr2'/Fm' im Licht) ermöglichte. Dieser neu etablierte Parameter war abhängig von der Funktionalität der Elektronentransportkette und der Temperatur. Im Gegensatz dazu war Fq'/Fm' hauptsächlich von der Lichtintensität abhängig. Unter kontrollierten Bedingungen korrelierte die Elektronentransportrate (ETR) basierend auf Fq'/Fm' mit der ETR, die aus CO2-Assimilationsmessungen berechnet wurde. In dieser Studie wurde ein ausreichend großer Datensatz einschließlich Spektralmessungen unter feldähnlichen Bedingungen gesammelt, um Faktoren zu identifizieren, die den Tages- und Jahreszeitengang der Photosynthese bestimmen. Gemäß der Lasso-Regressionsanalyse war Fq'/Fm' abhängig von der photosynthetisch aktiven Photonenflussdichte (PPFD) und den Spektralindizes. Das lineare Modell erklärte fast 50% der Varianz in Fq'/Fm', welche über zwei Vegetationsperioden gemessen wurden. Der zweite Parameter Fr2/Fm bzw. Fr2'/Fm' wurde stark durch die Temperatur und die Nutzpflanzenart bestimmt, z.B. unterschied sich winterharter Raps und Soja bei niedrigeren Temperaturen deutlich. Nur ein geringer Einfluss auf die gemessenen Parameter wurde für die verschiedenen Jahre, Tageszeiten und damit Saison- oder Pflanzenentwicklungsstadium festgestellt. Im Folgenden wurden genotypische Unterschiede am Parametermittelwert oder der Wechselwirkung der Parameter mit den Umweltfaktoren PPFD und Temperatur analysiert. Zunehmender Trockenstress verringerte die Fq'/Fm' in Mais. Im Gegensatz dazu stieg die Fr2/Fm bzw. Fr2'/Fm' als Reaktion auf Trockenheit an, was auf einen verstärkten zyklischen Elektronentransport hindeutet. Eine Infektion mit echtem Mehltau wurde durch Fq'/Fm' festgestellt, bevor die Symptome mit dem Auge sichtbar waren. Bei einer Anayse aller gesammelten Felddaten war Fq'/Fm' wieder abhängig von PPFD, korrelierte jedoch stärker mit der Reflexion von Sonnenlicht bei 685 nm auf dem gemessenen Blatt. Die Reaktion von Fr2/Fm bzw. Fr2'/Fm' auf die Temperatur war auch im Feld zu beobachten und erklärte 79% aller Varianz in Mais. Der LIFT-Screening-Ansatz identifizierte tolerante Genotypen bezüglich der Licht- und Temperaturnutzungseffizienz unter Kontroll- und Stressbedingungen. Die Interaktion der betrachteten Parameter in Bezug auf PPFD und Temperatur ermöglicht die Vorhersage und Optimierung der photosynthetischen Leistung unter verschiedenen Umweltbedingungen

NLR immune receptors and diverse types of non-NLR proteins control race-specific resistance in Triticeae

Recent progress in large-scale sequencing, genomics, and rapid gene isolation techniques has accelerated the identification of race-specific resistance (R) genes and their corresponding avirulence (Avr) genes in wheat, barley, rye, and their wild relatives. Here, we describe the growing repertoire of identified R and Avr genes with special emphasis on novel R gene architectures, revealing that there is a large diversity of proteins encoded by race-specific resistance genes that extends beyond the canonical nucleotide-binding domain leucine-rich repeat proteins. Immune receptors with unique domain architectures controlling race-specific resistance possibly reveal novel aspects on the biology of host-pathogen interactions. We conclude that the polyploid cereal genomes have a large evolutionary potential to generate diverse types of resistance genes

Development of simple sequence repeat markers specific for the Lr34 resistance region of wheat using sequence information from rice and Aegilops tauschii

Hexaploid wheat (Triticum aestivum L.) originated about 8,000years ago from the hybridization of tetraploid wheat with diploid Aegilops tauschii Coss. containing the D-genome. Thus, the bread wheat D-genome is evolutionary young and shows a low degree of polymorphism in the bread wheat gene pool. To increase marker density around the durable leaf rust resistance gene Lr34 located on chromosome 7DS, we used molecular information from the orthologous region in rice. Wheat expressed sequence tags (wESTs) were identified by homology with the rice genes in the interval of interest, but were monomorphic in the ‘Arina'בForno' mapping population. To derive new polymorphic markers, bacterial artificial chromosome (BAC) clones representing a total physical size of ∼1Mb and belonging to four contigs were isolated from Ae. tauschii by hybridization screening with wheat ESTs. Several BAC clones were low-pass sequenced, resulting in a total of ∼560kb of sequence. Ten microsatellite sequences were found, and three of them were polymorphic in our population and were genetically mapped close to Lr34. Comparative analysis of marker order revealed a large inversion between the rice genome and the wheat D-genome. The SWM10 microsatellite is closely linked to Lr34 and has the same allele in the three independent sources of Lr34: ‘Frontana', ‘Chinese Spring', and ‘Forno', as well in most of the genotypes containing Lr34. Therefore, SWM10 is a highly useful marker to assist selection for Lr34 in breeding programs worldwid

Specific patterns of changes in wheat gene expression after treatment with three antifungal compounds

The two fungicides azoxystrobin and fenpropimorph are used against powdery mildew and rust diseases in wheat (Triticum aestivumL). Azoxystrobin, a strobilurin, inhibits fungal mitochondrial respiration and fenpropimorph, a morpholin, represses biosynthesis of ergosterol, the major sterol of fungal membranes. Although the fungitoxic activity of these compounds is well understood, their effects on plant metabolism remain unclear. In contrast to the fungicides which directly affect pathogen metabolism, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methylester (BTH) induces resistance against wheat pathogens by the activation of systemic acquired resistance in the host plant. In this study, we monitored gene expression in spring wheat after treatment with each of these agrochemicals in a greenhouse trial using a microarray containing 600 barley cDNA clones. Defence-related genes were strongly induced after treatment with BTH, confirming the activation of a similar set of genes as in dicot plants following salicylic acid treatment. A similar gene expression pattern was observed after treatment with fenpropimorph and some defence-related genes were induced by azoxystrobin, demonstrating that these fungicides also activate a defence reaction. However, less intense responses were triggered than with BTH. The same experiments performed under field conditions gave dramatically different results. No gene showed differential expression after treatment and defence genes were already expressed at a high level before application of the agrochemicals. These differences in the expression patterns between the two environments demonstrate the importance of plant growth conditions for testing the impact of agrochemicals on plant metabolis

Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat

In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlr

Identification and genetic characterization of an Aegilops tauschii ortholog of the wheat leaf rust disease resistance gene Lr1

Aegilops tauschii (goat grass) is the progenitor of the Dgenome in hexaploid bread wheat. We have screened more than 200 Ae. tauschii accessions for resistance against leaf rust (Puccinia triticina) isolates, which are avirulent on the leaf rust resistance gene Lr1. Approximately 3.5% of the Ae. tauschii accessions displayed the same low infection type as the tester line Thatcher Lr1. The accession Tr.t.213, which showed resistance after artificial infection with Lr1 isolates both in Mexico and in Switzerland, was chosen for further analysis. Genetic analysis showed that the resistance in this accession is controlled by a single dominant gene, which mapped at the same chromosomal position as Lr1 in wheat. It was delimited in a 1.3-cM region between the restriction fragment length polymorphism (RFLP) markers ABC718 and PSR567 on chromosome5DL of Ae. tauschii. The gene was more tightly linked to PSR567 (0.47cM) than to ABC718 (0.79cM). These results indicate that the resistance gene in Ae. tauschii accession Tr.t.213 is an ortholog of the leaf rust resistance gene Lr1 of bread wheat, suggesting that Lr1 originally evolved in diploid goat grass and was introgressed into the wheat Dgenome during or after domestication of hexaploid wheat. Compared to hexaploid wheat, higher marker polymorphism and recombination frequencies were observed in the region of the Lr1 ortholog in Ae. tauschii. The identification of Lr1Ae, the orthologous gene of wheat Lr1, in Ae. tauschii will allow map-based cloning of Lr1 from this genetically simpler, diploid genom

Molecular approaches for characterization and use of natural disease resistance in wheat

Wheat production is threatened by a constantly changing population of pathogen species and races. Given the rapid ability of many pathogens to overcome genetic resistance, the identification and practical implementation of new sources of resistance is essential. Landraces and wild relatives of wheat have played an important role as genetic resources for the improvement of disease resistance. The use of molecular approaches, particularly molecular markers, has allowed better characterization of the genetic diversity in wheat germplasm. In addition, the molecular cloning of major resistance (R) genes has recently been achieved in the large, polyploid wheat genome. For the first time this allows the study and analysis of the genetic variability of wheat R loci at the molecular level and therefore, to screen for allelic variation at such loci in the gene pool. Thus, strategies such as allele mining and ecotilling are now possible for characterization of wheat disease resistance. Here, we discuss the approaches, resources and potential tools to characterize and utilize the naturally occurring resistance diversity in wheat. We also report a first step in allele mining, where we characterize the occurrence of known resistance alleles at the wheat Pm3 powdery mildew resistance locus in a set of 1,320 landraces assembled on the basis of eco-geographical criteria. From known Pm3 R alleles, only Pm3b was frequently identified (3% of the tested accessions). In the same set of landraces, we found a high frequency of a Pm3 haplotype carrying a susceptible allele of Pm3. This analysis allowed the identification of a set of resistant lines where new potentially functional alleles would be present. Newly identified resistance alleles will enrich the genetic basis of resistance in breeding programmes and contribute to wheat improvemen

Distribution of cadmium in leaves of Thlaspi caerulescens

Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 μM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cell

Host Adaptation Through Hybridization: Genome Analysis of Triticale Powdery Mildew Reveals Unique Combination of Lineage-Specific Effectors

The emergence of new fungal pathogens through hybridization represents a serious challenge for agriculture. Hybridization between the wheat mildew (Blumeria graminis f. sp. tritici) and rye mildew (B. graminis f. sp. secalis) pathogens has led to the emergence of a new mildew form (B. graminis f. sp. triticale) growing on triticale, a man-made amphiploid crop derived from crossing rye and wheat, which was originally resistant to the powdery mildew disease. The identification of the genetic basis of host adaptation in triticale mildew has been hampered by the lack of a reference genome. Here, we report the 141.4-Mb reference assembly of triticale mildew isolate THUN-12 derived from long-read sequencing and genetic map-based scaffolding. All 11 triticale mildew chromosomes were assembled from telomere-to-telomere and revealed that 19.7% of the hybrid genome was inherited from the rye mildew parental lineage. We identified lineage-specific regions in the hybrid, inherited from the rye or wheat mildew parental lineages, that harbor numerous bona fide candidate effectors. We propose that the combination of lineage-specific effectors in the hybrid genome is crucial for host adaptation, allowing the fungus to simultaneously circumvent the immune systems contributed by wheat and rye in the triticale crop. In line with this, we demonstrate the functional transfer of the SvrPm3 effector from wheat to triticale mildew, a virulence effector that specifically suppresses resistance of the wheat Pm3 allelic series. This transfer is the likely underlying cause for the observed poor effectiveness of several Pm3 alleles against triticale mildew and exemplifies the negative implications of pathogen hybridizations on resistance breeding. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license