Documentation system for plant transformation service and research

Background: In plant transformation, method compliance is critical for success. Transformation methods are complicated and tend to evolve over time. Until the complete method is published, method details are often partially orally transmitted and thus bound to a few people. Their documentation in text files are often a mixture of material and method description with many references to other sources especially to media description. These media are complex and often composed from several commercially available mixtures plus individually prepared stocks. The actual transformation experiment is generally documented in lab books, in which deviations from the methods and results are reported. Additionally, work schedules are planned in diaries. Both paper-based sources lack backup copies and miss unambiguous links to method descriptions and media recipes. Description: To solve the problem, we devised a standard-operation-procedure system based on a Microsoft Access database containing the interlinked modules 'Media', 'Methods' and 'Experiments'. The Media module contains all basic chemicals, stocks and complex media. In this module, complex media are composed from other elements of the Media module, thus mimicking the workflows of media preparation in the lab. The Media module is made attractive to the user by functions that generate file cards and labels. The Methods module describes each method stepwise and links the steps to the media. Copy functions allow cloning of old methods to document method evolution without alteration of the old methods. Activation and inactivation functions in the Media and the Methods module remove outdated entries from active use. The Experiments module links the method to experiment specific information. This module generates a lab-book like user interface and a work schedule, and it contains a simple result section. Conclusion: The system has been evolved and tested over several years in a transformation service unit, where it increased efficiency. Additionally, the system provided rapid access to data for quality control and decision making. The system can be easily modified for the use in other research environments

Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy

Defective α-galactosidase A (AGAL/GLA) due to missense or nonsense mutations in the GLA gene results in accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and its deacylated derivate globotriaosylsphingosine (lyso-Gb3) in cells and body fluids. The aberrant glycosphingolipid metabolism leads to a progressive lysosomal storage disorder, i. e. Fabry disease (FD), characterized by chronic inflammation leading to multiorgan damage. Enzyme replacement therapy (ERT) with agalsidase-alfa or -beta is one of the main treatment options facilitating cellular Gb3 clearance. Proteome studies have shown changes in complement proteins during ERT. However, the direct activation of the complement system during FD has not been explored. Here, we demonstrate strong activation of the complement system in 17 classical male FD patients with either missense or nonsense mutations before and after ERT as evidenced by high C3a and C5a serum levels. In contrast to the strong reduction of lyso-Gb3 under ERT, C3a and C5a markedly increased in FD patients with nonsense mutations, most of whom developed anti-drug antibodies (ADA), whereas FD patients with missense mutations, which were ADA-negative, showed heterogenous C3a and C5a serum levels under treatment. In addition to the complement activation, we found increased IL-6, IL-10 and TGF-ß1 serum levels in FD patients. This increase was most prominent in patients with missense mutations under ERT, most of whom developed mild nephropathy with decreased estimated glomerular filtration rate. Together, our findings demonstrate strong complement activation in FD independent of ERT therapy, especially in males with nonsense mutations and the development of ADAs. In addition, our data suggest kidney cell-associated production of cytokines, which have a strong potential to drive renal damage. Thus, chronic inflammation as a driver of organ damage in FD seems to proceed despite ERT and may prove useful as a target to cope with progressive organ damage

Expression profiling of rice cultivars differing in their tolerance to long-term drought stress

Understanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype × environment (G × E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G × E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G × E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars

A Naturally Associated Rhizobacterium of Arabidopsis thaliana Induces a Starvation-Like Transcriptional Response while Promoting Growth

Plant growth promotion by rhizobacteria is a known phenomenon but the underlying mechanisms are poorly understood. We searched for plant growth-promoting rhizobacteria that are naturally associated with Arabidopsis thaliana to investigate the molecular mechanisms that are involved in plant growth-promotion. We isolated a Pseudomonas bacterium (Pseudomonas sp. G62) from roots of field-grown Arabidopsis plants that has not been described previously and analyzed its effect on plant growth, gene expression and the level of sugars and amino acids in the host plant. Inoculation with Pseudomonas sp. G62 promoted plant growth under various growth conditions. Microarray analysis revealed rapid changes in transcript levels of genes annotated to energy-, sugar- and cell wall metabolism in plants 6 h after root inoculation with P. sp. G62. The expression of several of these genes remained stable over weeks, but appeared differentially regulated in roots and shoots. The global gene expression profile observed after inoculation with P. sp. G62 showed a striking resemblance with previously described carbohydrate starvation experiments, although plants were not depleted from soluble sugars, and even showed a slight increase of the sucrose level in roots 5 weeks after inoculation. We suggest that the starvation-like transcriptional phenotype - while steady state sucrose levels are not reduced - is induced by a yet unknown signal from the bacterium that simulates sugar starvation. We discuss the potential effects of the sugar starvation signal on plant growth promotion

Confounding Factors in Container-Based Drought Tolerance Assessments in Solanum tuberosum

Potato is an important food crop with high water-use-efficiency but low drought tolerance. The bottleneck in drought tolerance breeding is phenotyping in managed field environments. Fundamental research on drought tolerance is predominantly done in container-based test systems in controlled environments. However, the portability of results from these systems to performance under field conditions is debated. Thus, we analyzed the effects of climate conditions, container size, starting material, and substrate on yield and drought tolerance assessment of potato genotypes compared to field trials. A leave one out assessment indicated a minimum of three field trials for stable tolerance prediction. The tolerance ranking was highly reproducible under controlled-conditions, but weakly correlated with field performance. Changing to variable climate conditions, increasing container size, and substituting cuttings by seed tubers did not improve the correlation. Substituting horticultural substrate by sandy soil resulted in yield and tuber size distributions similar to those under field conditions. However, as the effect of the treatment × genotype × substrate interaction on yield was low, drought tolerance indices that depend on relative yields can be assessed on horticultural substrate also. Realistic estimates of tuber yield and tuber size distribution, however, require the use of soil-based substrates

SAS scripts for evalution of phenotyping and metabolite data

<p>This archive file contains SAS-scripts for the evaluation of phenotyping and metabolite data gained on Arabidopsis plants grown under different light qualities. The use  of LED systems for growing Arabidopsis thaliana under controlled conditions raises concerns as to the reproducibility of data gained with plants grown under fluorescent lamps. We compared growth, development and reproduction as well as photosynthetic parameters and metabolite content in different Arabidopsis genotypes grown under two LED systems or fluorescent lamps. We came to the conclusion that the effect caused by the change to LED quality is small compared to the effects of plant age and diurnal rhythm.</p

Finding Phenotypic Biomarkers for Drought Tolerance in <i>Solanum tuberosum</i>

Climate change models predict increased drought frequencies. Maintaining yield stability necessitates drought-tolerant crops. However, their breeding is challenging; drought tolerance is a multigene trait with significant environment interaction. Thus, the training of genomic selection models requires phenotyping a large genotype population under arid conditions. We aimed to identify phenotypic tolerance traits that facilitate the screening of large populations in the field. We performed three trials on 20 tetraploid Solanum tuberosum ssp. tuberosum genotypes with significant drought tolerance variation. Plants were subjected to early, late and long-term drought under variable climate conditions. For each stress scenario, the drought tolerance index DRYMp was calculated from the relative tuber starch yield. A laser scanner system measured canopy development continuously over the crop’s lifecycle and provided estimates of leaf movement and canopy growth features. Growth curves were evaluated by logistic regression. Different multiple regression approaches were compared for their ability to predict tolerance from phenotype data of optimally watered or stressed plants. We established that early short-term stress can be used as a proxy for long-term stress in the absence of genetic variation for drought stress recovery or memory. The gen-otypes varied significantly in most canopy features. Leaf-area-based features combined significant genotype effects with environmental stability. Multiple regression models based on single-day data outperformed those based on the regression curve parameter. The models included leaf area and leaf position parameters and partially reproduced prior findings on siblings in a genetically more diverse population