Asymmetric distribution of extracellular matrix proteins in seed coat epidermal cells of Arabidopsis is determined by polar secretion.

Although asymmetric deposition of the plant extracellular matrix is critical for the normal functioning of many cell types, the molecular mechanisms establishing this asymmetry are not well understood. During differentiation, Arabidopsis seed coat epidermal cells deposit large amounts of pectin-rich mucilage asymmetrically to form an extracellular pocket between the plasma membrane and the outer tangential primary cell wall. At maturity, the mucilage expands on contact with water, ruptures the primary cell wall, and extrudes to encapsulate the seed. In addition to polysaccharides, mucilage contains secreted proteins including the ß-galactosidase MUCILAGE MODIFIED 2 (MUM2). A functional chimeric protein where MUM2 was fused translationally with Citrine yellow fluorescent protein (Citrine) indicated that MUM2-Citrine fluorescence preferentially accumulates in the mucilage pocket concomitant with mucilage deposition and rapidly disappears when mucilage synthesis ceases. A secreted form of Citrine, secCitrine, showed a similar pattern of localization when expressed in developing seed coat epidermal cells. This result suggested that both the asymmetric localization and rapid decrease of fluorescence is not unique to MUM2-Citrine and may represent the default pathway for secreted proteins in this cell type. v-SNARE proteins were localized only in the membrane adjacent to the mucilage pocket, supporting the hypothesis that the cellular secretory apparatus is redirected and targets secretion to the outer periclinal apoplast during mucilage synthesis. In addition, mutation of ECHIDNA, a gene encoding a TGN-localized protein involved in vesicle targeting, causes misdirection of mucilage, MUM2 and v-SNARE proteins from the apoplast/plasma membrane to the vacuole/tonoplast. Western blot analyses suggested that the disappearance of MUM2-Citrine fluorescence at the end of mucilage synthesis is due to protein degradation and because several proteases have been identified in extruded seed mucilage. However, as mutation of these genes did not result in a substantial delay in MUM2-Citrine degradation and the timing of their expression and/or their intracellular localization were not consistent with a role in MUM2-Citrine disappearance, the mechanism underlying the abrupt decrease of MUM2-Citrine remains unclear.

Expression Patterns and Functional Characterization of Arabidopsis Galactose Oxidase-Like Genes Suggest Specialized Roles for Galactose Oxidases in Plants.

Galactose oxidases (GalOxs) are well-known enzymes that have been identified in several fungal species and characterized using structural and enzymatic approaches. However, until very recently, almost no information on their biological functions was available. The Arabidopsis (Arabidopsis thaliana) gene ruby particles in mucilage (RUBY) encodes a putative plant GalOx that is required for pectin cross-linking through modification of galactose (Gal) side chains and promotes cell-cell adhesion between seed coat epidermal cells. RUBY is one member of a family of seven putative GalOxs encoded in the Arabidopsis genome. To examine the function(s) of GalOxs in plants, we studied the remaining six galactose oxidase-like (GOXL) proteins. Like RUBY, four of these proteins (GOXL1, GOXL3, GOXL5 and GOXL6) were found to localize primarily to the apoplast, while GOXL2 and GOXL4 were found primarily in the cytoplasm. Complementation and GalOx assay data suggested that GOXL1, GOXL3 and possibly GOXL6 have similar biochemical activity to RUBY, whereas GOXL5 only weakly complemented and GOXL2 and GOXL4 showed no activity. Members of this protein family separated into four distinct clades prior to the divergence of the angiosperms. There have been recent duplications in Brassicaceae resulting in two closely related pairs of genes that have either retained similarity in expression (GOXL1 and GOXL6) or show expression divergence (GOXL3 and RUBY). Mutant phenotypes were not detected when these genes were disrupted, but their expression patterns suggest that these proteins may function in tissues that require mechanical reinforcements in the absence of lignification.

RUBY, a Putative Galactose Oxidase, Influences Pectin Properties and Promotes Cell-To-Cell Adhesion in the Seed Coat Epidermis of Arabidopsis.

Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis (Arabidopsis thaliana) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE (RUBY), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUBY is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 (mum2) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains.

A mutant Brassica napus (canola) population for the identification of new genetic diversity via TILLING and next generation sequencing.

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.

Reverse genetics techniques: engineering loss and gain of gene function in plants.

Genetic analysis represents a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo. Genome sequencing projects have identified large numbers of plant genes for which no role has yet been defined. To address this problem a number of techniques have been developed, over the last 15 years, to enable researchers to identify plants with mutations in genes of known sequence. These reverse genetic approaches include RNAi and related technologies and screening of populations mutagenised by insertion (PCR), deletion (PCR) and point mutation (TILLING), each with its own strengths and weaknesses. The development of next-generation sequencing techniques now allows such screening to be done by sequencing. In the future, it is likely that the genomes of thousands of plants from mutagenised populations will be sequenced allowing for the identification of plants with mutations in specific genes to be done in silico.

Forward and reverse genetics of rapid-cycling Brassica oleracea.

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today's B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes.

TILLING is an effective reverse genetics technique for Caenorhabditis elegans.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified. RESULTS: To determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms. CONCLUSION: We conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa.

Abstract Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.

TILLING without a plough: a new method with applications for reverse genetics.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms. Its advantages over other reverse genetic techniques include its applicability to virtually any organism, its facility for high-throughput and its independence of genome size, reproductive system or generation time. TILLING is currently being used for the detection of both induced and natural variation in several plant species.

Improved detection of small deletions in complex pools of DNA.

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.