Large-scale Mutant Screens Research Articles

LBD29-Involved Auxin Signaling Represses NAC Master Regulators and Fiber Wall Biosynthesis.

NAM, ATAF1/2 and CUC2 (NAC) domain transcription factors function as master switches in regulating secondary cell wall (SCW) biosynthesis in Arabidopsis (Arabidopsis thaliana) stems. Despite the importance of these NACs in fiber development, the upstream signal is still elusive. Using a large-scale mutant screening, we identified a dominant activation-tagging mutant, fiberless-d (fls-d), showing defective SCW development in stem fibers, similar to that of the nac secondary wall thickening promoting factor1-1 (nst1-1)nst3-3 double mutant. Overexpression of LATERAL ORGAN BOUNDARIES DOMAIN29 (LBD29) is responsible for the fls-d mutant phenotypes. By contrast, loss-of-function of LBD29, either in the dominant repression transgenic lines or in the transfer-DNA (T-DNA) insertion mutant lbd29-1, enhanced SCW development in fibers. Genetic analysis and transgenic studies demonstrated LBD29 depends on master regulators in mediating SCW biosynthesis, specifically NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, and NST3. Increasing indole-3-acetic acid (IAA) levels, either in stem tissues above a N-1-naphthylphthalamic acid-treated region or in plants directly sprayed with IAA, inhibits fiber wall thickening. The inhibition effect of naphthylphthalamic acid treatment and exogenous IAA application depends on a known auxin signaling pathway involving AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and LBD29. These results demonstrate auxin is upstream of LBD29 in repressing NAC master regulators, and therefore shed new light on the regulation of SCW biosynthesis in Arabidopsis.

High-Throughput Analysis of Arabidopsis Stem Vibrations to Identify Mutants With Altered Mechanical Properties.

Mechanical properties are rarely used as quantitative indices for the large-scale mutant screening of plants, even in the model plant Arabidopsis thaliana. The mechanical properties of plant stems generally influence their vibrational characteristics. Here, we developed Python-based software, named AraVib, for the high-throughput analysis of free vibrations of plant stems, focusing specifically on Arabidopsis stem vibrations, and its extended version, named AraVibS, to identify mutants with altered mechanical properties. These programs can be used without knowledge of Python and require only an inexpensive handmade setting stand and an iPhone/iPad with a high-speed shooting function for data acquisition. Using our system, we identified an nst1 nst3 double-mutant lacking secondary cell walls in fiber cells and a wrky12 mutant displaying ectopic formation of secondary cell wall compared with wild type by employing only two growth traits (stem height and fresh weight) in addition to videos of stem vibrations. Furthermore, we calculated the logarithmic decrement, the damping ratio, the natural frequency and the stiffness based on the spring-mass-damper model from the video data using AraVib. The stiffness was estimated to be drastically decreased in nst1 nst3, which agreed with previous tensile test results. However, in wrky12, the stiffness was significantly increased. These results demonstrate the effectiveness of our new system. Because our method can be applied in a high-throughput manner, it can be used to screen for mutants with altered mechanical properties.

Nitrite is the driver, phytohormones are modulators while NO and H2O2 act as promoters of NO2-induced cell death.

This study aimed to understand the molecular mechanisms of nitrogen dioxide (NO2)-induced toxicity and cell death in plants. Exposure of Arabidopsis to high concentrations of NO2 induced cell death in a dose-dependent manner. No leaf symptoms were visible after fumigation for 1 h with 10 parts per million (ppm) NO2 However, 20 ppm NO2 caused necrotic lesion formation and 30 ppm NO2 complete leaf collapse, which had already started during the 1 h fumigation period. NO2 fumigation resulted in a massive accumulation of nitrite and in protein modifications by S-nitrosylation and tyrosine nitration. Nitric oxide (NO) at 30 ppm did not trigger leaf damage or any of the effects observed after NO2 fumigation. The onset of NO2-induced cell death correlated with NO and hydrogen peroxide (H2O2) signaling and a decrease in antioxidants. NO- and H2O2-accumulating mutants were more sensitive to NO2 than wild-type plants. Accordingly, experiments with specific scavengers confirmed that NO and H2O2 are essential promoters of NO2-induced cell death. Leaf injection of 100 mM nitrite caused an increase in S-nitrosylation, NO, H2O2, and cell death suggesting that nitrite functioned as a mediator of NO2-induced effects. A targeted screening of phytohormone mutants revealed a protective role of salicylic acid (SA) signaling in response to NO2 It was also shown that phytohormones were modulators rather than inducers of NO2-induced cell death. The established experimental set-up is a suitable system to investigate NO2 and cell death signaling in large-scale mutant screens.

Mutagenic effects of carbon ion beam irradiations on dry Lotus japonicus seeds

Carbon ion beam irradiation is a powerful method for creating mutants and has been used in crop breeding more and more. To investigate the effects of carbon ion beams on Lotus japonicus, dry seeds were irradiated by 80MeV/u carbon ion beam at dosages of 0, 100, 200, 300, 400, 500 and 600Gy. The germination rate, survival rate and root length of M1 populations were explored and the dose of 400Gy was selected as the median lethal dose (LD50) for a large-scale mutant screening. Among 2472 M2 plants, 127 morphological mutants including leaf, stem, flower and fruit phenotypic variation were found, and the mutation frequency was approximately 5.14%. Inter simple sequence repeat (ISSR) assays were utilized to investigate the DNA polymorphism between seven mutants and eight plants without phenotypic variation from M2 populations. No remarkable differences were detected between these two groups, and the total polymorphic rate was 0.567%.

A combination of gene expression ranking and co-expression network analysis increases discovery rate in large-scale mutant screens for novel Arabidopsis thaliana abiotic stress genes.

As challenges to food security increase, the demand for lead genes for improving crop production is growing. However, genetic screens of plant mutants typically yield very low frequencies of desired phenotypes. Here, we present a powerful computational approach for selecting candidate genes for screening insertion mutants. We combined ranking of Arabidopsis thaliana regulatory genes according to their expression in response to multiple abiotic stresses (Multiple Stress [MST] score), with stress-responsive RNA co-expression network analysis to select candidate multiple stress regulatory (MSTR) genes. Screening of 62 T-DNA insertion mutants defective in candidate MSTR genes, for abiotic stress germination phenotypes yielded a remarkable hit rate of up to 62%; this gene discovery rate is 48-fold greater than that of other large-scale insertional mutant screens. Moreover, the MST score of these genes could be used to prioritize them for screening. To evaluate the contribution of the co-expression analysis, we screened 64 additional mutant lines of MST-scored genes that did not appear in the RNA co-expression network. The screening of these MST-scored genes yielded a gene discovery rate of 36%, which is much higher than that of classic mutant screens but not as high as when picking candidate genes from the co-expression network. The MSTR co-expression network that we created, AraSTressRegNet is publicly available at http://netbio.bgu.ac.il/arnet. This systems biology-based screening approach combining gene ranking and network analysis could be generally applicable to enhancing identification of genes regulating additional processes in plants and other organisms provided that suitable transcriptome data are available.

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ (max)=390 nm) is converted to a red product (λ (max)=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A (390)/A (486) ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L(-1) and 50 μg L(-1) to 10 mg L(-1), respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.

Ethylmethanesulfonate (EMS) mutagenesis of Solanum lycopersicum cv. Micro-Tom for large-scale mutant screens

Sequencing a whole genome, cataloguing full-length cDNAs and ESTs, and construction of a comprehensive mutant population are essential steps in genome projects of individual model species. Tomato is one of the most important model crops to be undertaken for study over the next few decades, and the Solanaceae Genomics Project (SOL) has begun genome projects as international collaborations. Solanum lycopersicum cultivar Micro-Tom shares several advantages with Arabidopsis, including its small size, short life cycle (approximately 90 days), and growth normally under artificial light. Here we demonstrate an initial attempt to generate a comprehensive mutant population in Micro-Tom. A total of 3,839 M2 families derived from ethylmethanesulfonate (EMS) mutagenesis were visually phenotyped, and putative mutants were classified into 15 primary and 48 secondary categories based on the SOL database, The Genes That Make Tomatoes.

Transcriptome comparison of winter and spring wheat responding to low temperature

Freezing tolerance in plants is a complex trait that occurs in many plant species during growth at low, nonfreezing temperatures, a process known as cold acclimation. This process is regulated by a multigenic system expressing broad variation in the degree of freezing tolerance among wheat cultivars. Microarray analysis is a powerful and rapid approach to gene discovery. In species such as wheat, for which large scale mutant screening and transgenic studies are not currently practical, genotype comparison by this methodology represents an essential approach to identifying key genes in the acquisition of freezing tolerance. A microarray was constructed with PCR amplified cDNA inserts from 1184 wheat expressed sequence tags (ESTs) that represent 947 genes. Gene expression during cold acclimation was compared in 2 cultivars with marked differences in freezing tolerance. Transcript levels of more than 300 genes were altered by cold. Among these, 65 genes were regulated differently between the 2 cultivars for at least 1 time point. These include genes that encode potential regulatory proteins and proteins that act in plant metabolism, including protein kinases, putative transcription factors, Ca2+ binding proteins, a Golgi localized protein, an inorganic pyrophosphatase, a cell wall associated hydrolase, and proteins involved in photosynthesis.

Vertebrate model systems in the study of early heart development:Xenopus and zebrafish

Xenopus and zebrafish serve as outstanding models in which to study vertebrate heart development. The embryos are transparent, allowing observation during organogenesis; they can be obtained in large numbers; and they are readily accessible to embryologic manipulation and microinjection of RNA, DNA, or protein. These embryos can live by diffusion for several days, allowing analysis of mutants or experimental treatments that perturb normal heart development. Xenopus embryos have been used to understand the induction of the cardiac field, the role of Nkx genes in cardiac development, and the role transforming growth factor beta molecules in the establishment and signaling of left-right axis information. Large-scale mutant screens in zebrafish and the development of transgenics in both Xenopus and zebrafish have accelerated the molecular identification of genes that regulate conserved steps in cardiovascular development.

Genetics of neural development in zebrafish

Large-scale mutant screens in zebrafish have led to the identification of more than 50 genes affecting various aspects of neural development and function, including neural induction, anteroposterior and dorsoventral regionalization, axon pathfinding, neuronal differentiation and survival, and behavior. Phenotypic analysis of mutants for some of these genes has already uncovered important genetic and cellular interactions during development. Ongoing molecular analyses promise to further elucidate the mechanisms underlying neural development in vertebrates.

Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor.

According to a model based on embryological studies in amphibia, dorsoventral patterning is regulated by the antagonizing function of ventralizing bone morphogenetic proteins (BMPs) and dorsalizing signals generated by Spemann's organizer. Large-scale mutant screens in the zebrafish, Danio rerio, have led to the isolation of two classes of recessive lethal mutations affecting early dorsoventral pattern formation. dino mutant embryos are ventralized, whereas swirl mutants are dorsalized. We show that at early gastrula stages, dino and swirl mutants display an expanded or reduced Bmp4 expression, respectively. The dino and swirl mutant phenotypes both can be phenocopied and rescued by the modulation of BMP signaling in wild-type and mutant embryos. By suppressing BMP signaling in dino mutants, adult fertile dino -/- fish were generated. These findings, together with results from the analysis of dino-swirl double mutants, indicate that dino fulfills its dorsalizing activity via a suppression of swirl-dependent, BMP-like ventralizing activities. Finally, cell transplantation experiments show that dino is required on the dorsal side of early gastrula embryos and acts in a non-cell-autonomous fashion. Together, these results provide genetic evidence in support of a mechanism of early dorsoventral patterning that is conserved among vertebrate and invertebrate embryos.

A new paradigm for operant conditioning of Drosophila melanogaster.

A freely walking single fly (Drosophila melanogaster) can be conditioned to avoid one side of a small test chamber if the chamber is heated whenever the fly enters this side. In a subsequent memory test without heat it keeps avoiding the heat-associated side. The memory mutants dunce and rutabaga successfully avoid the heated side but show no avoidance in the memory test. Wildtype flies can be trained to successively avoid alternating sides in a reversal conditioning experiment. Every single fly shows strong avoidance and a positive memory score. The new conditioning apparatus has several advantages: (1) Statistically significant learning scores can be obtained for individual flies. (2) Learning scores are obtained fully automatically without interference of the experimenter. (3) The procedure is fast, robust and requires little handling. Therefore the apparatus is suitable for largescale mutant screening. (4) Animals are not attached to a hook and thus can easily be used for breeding.