Tomato growth promotion by the fungal endophytes Serendipita indica and Serendipita herbamans is associated with sucrose de-novo synthesis in roots and differential local and systemic effects on carbohydrate metabolisms and gene expression.

Plant growth-promoting and stress resilience-inducing root endophytic fungi represent an additional carbohydrate sink. This study aims to test if such root endophytes affect the sugar metabolism of the host plant to divert the flow of resources for their purposes. Fresh and dry weights of roots and shoots of tomato (Solanum lycopersicum) colonised by the closely related Serendipita indica and Serendipita herbamans were recorded. Plant carbohydrate metabolism was analysed by measuring sugar levels, by determining activity signatures of key enzymes of carbohydrate metabolism, and by quantifying mRNA levels of genes involved in sugar transport and turnover. During the interaction with the tomato plants, both fungi promoted root growth and shifted shoot biomass from stem to leaf tissues, resulting in increased leaf size. A common effect induced by both fungi was the inhibition of phosphofructokinase (PFK) in roots and leaves. This glycolytic-pacing enzyme shows how the glycolysis rate is reduced in plants and, eventually, how sugars are allocated to different tissues. Sucrose phosphate synthase (SPS) activity was strongly induced in colonised roots. This was accompanied by increased SPS-A1 gene expression in S. herbamans-colonised roots and by increased sucrose amounts in roots colonised by S. indica. Other enzyme activities were barely affected by S. indica, but mainly induced in leaves of S. herbamans-colonised plants and decreased in roots. This study suggests that two closely related root endophytic fungi differentially influence plant carbohydrate metabolism locally and systemically, but both induce a similar increase in plant biomass. Notably, both fungal endophytes induce an increase in SPS activity and, in the case of S. indica, sucrose resynthesis in roots. In leaves of S. indica-colonised plants, SWEET11b expression was enhanced, thus we assume that excess sucrose was exported by this transporter to the roots. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.

Enzyme activity profiling for physiological phenotyping within functional phenomics: plant growth and stress responses.

High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualization tools within a case study. This includes the introduction of multivariate statistical analyses that can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications in both basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops.

The scope of flavin-dependent reactions and processes in the model plant Arabidopsis thaliana.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized as coenzymes in many biochemical reduction-oxidation reactions owing to the ability of the tricyclic isoalloxazine ring system to employ the oxidized, radical and reduced state. We have analyzed the genome of Arabidopsis thaliana to establish an inventory of genes encoding flavin-dependent enzymes (flavoenzymes) as a basis to explore the range of flavin-dependent biochemical reactions that occur in this model plant. Expectedly, flavoenzymes catalyze many pivotal reactions in primary catabolism, which are connected to the degradation of basic metabolites, such as fatty and amino acids as well as carbohydrates and purines. On the other hand, flavoenzymes play diverse roles in anabolic reactions most notably the biosynthesis of amino acids as well as the biosynthesis of pyrimidines and sterols. Importantly, the role of flavoenzymes goes much beyond these basic reactions and extends into pathways that are equally crucial for plant life, for example the production of natural products. In this context, we outline the participation of flavoenzymes in the biosynthesis and maintenance of cofactors, coenzymes and accessory plant pigments (e. g. carotenoids) as well as phytohormones. Moreover, several multigene families have emerged as important components of plant immunity, for example the family of berberine bridge enzyme-like enzymes, flavin-dependent monooxygenases and NADPH oxidases. Furthermore, the versatility of flavoenzymes is highlighted by their role in reactions leading to tRNA-modifications, chromatin regulation and cellular redox homeostasis. The favorable photochemical properties of the flavin chromophore are exploited by photoreceptors to govern crucial processes of plant adaptation and development. Finally, a sequence- and structure-based approach was undertaken to gain insight into the catalytic role of uncharacterized flavoenzymes indicating their involvement in unknown biochemical reactions and pathways in A. thaliana.

Early-stage sugar beet taproot development is characterized by three distinct physiological phases.

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.

Simple and robust determination of the activity signature of key carbohydrate metabolism enzymes for physiological phenotyping in model and crop plants.

The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance, crop yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric kinetic assays. The comparison of extraction buffers and requirement for dialysis of crude protein extracts resulted in a universal protein extraction protocol, suitable for the preparation of protein extracts from different organs of various species. Individual published kinetic activity assays were optimized and adapted for a semi-high-throughput 96-well assay format. These assays proved to be robust and are thus suitable for physiological phenotyping, enabling the characterization and diagnosis of the physiological state. The potential of the determination of distinct enzyme activity signatures as part of a physiological fingerprint was shown for various organs and tissues from three monocot and five dicot model and crop species, including two case studies with external stimuli. Differential and specific enzyme activity signatures are apparent during inflorescence development and upon in vitro cold treatment of young inflorescences in the monocot ryegrass, related to conditions for doubled haploid formation. Likewise, treatment of dicot spring oilseed rape with elevated CO2 concentration resulted in distinct patterns of enzyme activity responses in leaves.