Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly ...show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.
Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their ...potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of
(susceptible, mildew locus o
and Mildew locus a
resistant). Multispectral imaging of barley plants was daily conducted 0-8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with
and
based resistances 0-120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of
f.sp.
conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The
resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening.
The infection of plants with pathogens results in the induction of defence reactions as well as changes in carbohydrate metabolism. On the one hand, the pathogen attempts to manipulate the ...carbohydrate metabolism of the plant for its own advantage. On the other, the plant has to reorganize carbon fluxes to ensure fight against the pathogen. In order to further investigate the connection between pathogen infection and carbohydrate metabolism, the effects of two types of pathogen, biotrophic and necrotrophic, on gene expression, endogenous sugar levels and photosynthesis of tomato plants were analysed. Photosynthetic gene expression was downregulated on infection with Pseudomonas syringae and Botrytis cinerea. In contrast, expression of a sink‐specific gene encoding a cell wall invertase and of defence genes was induced by both pathogens. These results provide evidence for a co‐regulation of defence, sink and photosynthetic gene expression in planta in response to both types of pathogen. The brassinosteroid‐containing plant restorative ComCat enhanced resistance against B. cinerea and counter‐regulated the repression of photosynthetic gene expression. Endogenous sugar levels decreased and the hexose to sucrose ratio increased on treatment with B. cinerea. The application of chlorophyll fluorescence imaging revealed the spatio‐temporal heterogeneity of the pathogen response. At 24 h after infection, inhibition of photosynthetic electron transport was restricted to the direct vicinity of the infection site, which was surrounded by a circle of increased photosynthetic activity. The photosynthesis of the remaining leaf was not affected at this stage. These results show the usefulness of chlorophyll fluorescence imaging for the assessment of the complex spatio‐temporal changes and for the definition of the areas relevant for other types of determination, e.g. gene expression.
One of the major challenges in agriculture is to ensure sufficient and healthy food availability for the increasing world population in near future. This requires maintaining sustainable cultivation ...of crop plants under varying environmental stresses. Among these stresses, salinity is the second most abundant threat worldwide after drought. One of the promising strategies to mitigate salinity stress is to cultivate halotolerant crops such as quinoa. Under high salinity, performance can be improved by plant growth promoting bacteria (PGPB). Among PGPB, endophytic bacteria are considered better in stimulating plant growth compared to rhizosphere bacteria because of their ability to colonize both in plant rhizosphere and plant interior. Therefore, in the current study, a pot experiment was conducted in a controlled greenhouse to investigate the effects of endophytic bacteria i.e.,
PsJN on improving growth, physiology and yield of quinoa under salinity stress. At six leaves stage, plants were irrigated with saline water having either 0 (control) or 400 mM NaCl. The results indicated that plants inoculated with PsJN mitigated the negative effects of salinity on quinoa resulting in increased shoot biomass, grain weight and grain yield by 12%, 18% and 41% respectively, over un-inoculated control. Moreover, inoculation with PsJN improved osmotic adjustment and ion homeostasis ability. In addition, leaves were also characterized for five key reactive oxygen species (ROS) scavenging enzyme in response to PsJN treatment. This showed higher activity of catalase (CAT) and dehydroascobate reductase (DHAR) in PsJN-treated plants. These findings suggest that inoculation of quinoa seeds with
PsJN could be used for stimulating growth and yield of quinoa in highly salt-affected soils.
Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with '
Phytoplasma solani', but molecular interactions between the causal pathogen and its host plant are ...not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with '
. P. solani' in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.
Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins ...that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.
Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples ...is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E).
Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.
Despite the agronomic importance of sugar beet (Beta vulgaris L.), the early‐stage development of its taproot has only been poorly investigated. Thus, the mechanisms that determine growth and sugar ...accumulation in sugar beet are largely unknown. In the presented study, a physiological characterization of early‐stage sugar beet taproot development was conducted. Activities were analyzed for fourteen key enzymes of carbohydrate metabolism in developing taproots over the first 80 days after sowing. In addition, we performed in situ localizations of selected carbohydrate‐metabolic enzyme activities, anatomical investigations, and quantifications of soluble carbohydrates, hexose phosphates, and phytohormones. Based on the accumulation dynamics of biomass and sucrose, as well as on anatomical parameters, the early phase of taproot development could be subdivided into three stages—prestorage, transition, secondary growth and sucrose accumulation stage—each of which was characterized by distinct metabolic and phytohormonal signatures. The enzyme activity signatures corresponding to these stages were also shown to be robustly reproducible in experiments conducted in two additional locations. The results from this physiological phenotyping approach contribute to the identification of the key regulators of sugar beet taproot development and open up new perspectives for sugar beet crop improvement concerning both physiological marker‐based breeding and biotechnological approaches.
The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘
...Candidiatus
Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘
Ca
. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing
Nicotiana benthamiana
leaves after
Agrobacterium
-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.
Phenotyping in field experiments is challenging due to interactions between plants and effects from biotic and abiotic factors which increase complexity in plant development. In such environments, ...visual or destructive measurements are considered the limiting factor and novel approaches are necessary. Remote multispectral imaging is a powerful method that has shown significant potential to estimate crop physiology. However, precise measurements of phenotypic differences between crop varieties in field experiments require exclusion of the disturbances caused by wind and varying sunlight. A mobile and closed multispectral imaging system was developed to study canopies in field experiments. This system shuts out wind and sunlight to ensure the highest possible precision and accuracy. Multispectral images were acquired in an experiment with four different wheat varieties, two different nitrogen levels, replicated on two different soil types at four different dates from 15 May (BBCH 13) to 18 June (BBCH 41 to 57). The images were analyzed and derived vegetation coverage and Normalized Difference Vegetation index (NDVI) were used to assess varietal differences. The results showed potentials for differentiating between the varieties using both vegetation coverage and NDVI, especially at the early growth stages. The perspectives of high-precision and high-throughput imaging for field phenotyping are discussed including the potentials of measuring varietal differences via spectral imaging in comparison to other simpler technologies such as spectral reflectance and RGB imaging.
PDF
