No abstract

Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.

Plant development represents a continuous process in which the plant undergoes morphological, (epi)genetic and metabolic changes. Starting from pollination, seed maturation and germination, the plant continues to grow and develops specialized organs to survive, thrive and generate offspring. The development of plants and the interplay with its environment are highly linked to glycosylation of proteins and lipids as well as metabolism and signaling of sugars. Although the involvement of these protein modifications and sugars is well-studied, there is still a long road ahead to profoundly comprehend their nature, significance, importance for plant development and the interplay with stress responses. This review, approached from the plants’ perspective, aims to focus on some key findings highlighting the importance of glycosylation and sugar signaling for plant development.

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases only hydrolyse terminal Fru residues (fructan exohydrolase, FEH). Levan (β-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (β-2,1 linkages), harbouring several 1-FEH isoforms for their degradation. Here, a novel chicory fructanase with high affinity for levan was characterized, providing evidence that such enzymes widely occur in higher plants. It is adapted to common microbial fructan profiles, but has low affinity towards chicory inulin, in line with a function in trimming of microbial fructans in the extracellular environment. Docking experiments indicate the importance of an N-glycosylation site close to the active site for substrate specificity. Optimal pH and temperature for levan hydrolysis are 5.0 and 43.7°C, respectively. Docking experiments suggested multiple substrate binding sites and levan-mediated enzyme dimerization, explaining the observed positive cooperativity (Hill kinetics). Alignments show a one amino acid shift in the position of a conserved DXX(R/K) couple, typical for sucrose binding in cell wall invertases. A possible involvement of plant fructanases in levan trimming is discussed, in line with the emerging “fructan detour” concepts, suggesting that levan oligosaccharides act as signalling entities during plant microbial interactions.

To date, little attention has been paid to the genotypic plasticity and influence of the fermentation process on gene functions and biological processes in cacao beans. The primary tools for such analyses are gene expression studies with reverse transcription quantitative PCR (RT-qPCR). While this is a well-appreciated technique, it is only reliable when considering the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, which is unfortunately barely applied in plant sciences and non-existent in cacao-related studies. In this study, an appropriate from bean to RT-qPCR protocol was developed. In total, sixty-five candidate reference gene (RG) assays were validated. These assays were either adopted from literature (traditional "housekeeping" genes) or based on RNA-sequencing data (novel). After validation, three novel reference genes (SUGP1, NAP1, SGT1) were recommended for normalization of gene expression within fermented cacao beans. The suitability of the novel candidates surpassed the traditional housekeeping genes. In addition, these assays seemed largely unaffected by RNA integrity. This is the first study to establish a standardized RT-qPCR workflow on cacao beans during fermentation, facilitating future studies. We recommend similar MIQE-based approaches for future gene expression studies on other organisms for miscellaneous objectives.