Tracey Welham

A TILLING Reverse Genetics Tool and a Web-Accessible Collection of Mutants of the Legume Lotus japonicus

Reverse genetics aims to identify the function of a gene with known sequence by phenotypic analysis of cells or organisms in which the function of this gene is impaired. Commonly used strategies for reverse genetics encompass transposon mutagenesis (Tissier et al., 1999) and RNA-mediated gene silencing or RNA interference (Voinnet, 2002). We adopted a complementary strategy to set up a reverse genetics tool for the legume Lotus japonicus that identifies individuals carrying point mutations in any gene of interest within a large population of ethyl methanesulfonate (EMS)-mutagenized M2 plants. This strategy was first described by McCallum et al. (2000a,b) using the acronym TILLING (Targeted Induced Local Lesions in Genomes). The target sequence is PCR amplified from pooled M2 individuals. DNA with point mutations are detected by melting and reannealing of the PCR products. This results in the formation of heteroduplex DNA in which one strand originates from the mutant and the other from the wild-type PCR product. A mismatch occurs at the site of the point mutation, which can be detected using mismatch-specific endonucleases such as CEL I from celery (Apium graveolens; Yang et al., 2000). This enzyme recognizes mismatches in heteroduplex DNA and cleaves DNA specifically at the mismatched site. The cleavage products can be separated by gel electrophoresis, typically sequencing-type denaturing PAGE. This method of mismatch detection is amenable to pooling strategies. In the Arabidopsis TILLING facility, DNA of eight M2 plants is mixed to form a pool (Colbert et al., 2001). At this pool size, a population of 768 individuals can be screened by PCR in a 96-well microtiter plate, and run on one 96-well gel, each well representing eight individuals. Individuals from pools yielding cleavage products are then PCR amplified individually to identify the mutation bearing plant, progeny of which will segregate the mutation of interest.

TILLING Mutants of Lotus japonicus Reveal That Nitrogen Assimilation and Fixation Can Occur in the Absence of Nodule-Enhanced Sucrose Synthase

In all plant species studied to date, sucrose synthase occurs as multiple isoforms. The specific functions of the different isoforms are for the most part not clear. Six isoforms of sucrose synthase have been identified in the model legume Lotus japonicus, the same number as in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The genes encoding these isoforms are differentially expressed in all plant organs examined, although one, LjSUS4, is only expressed in flowers. LjSUS1 is the most highly expressed in all plant organs tested, except root nodules, where LjSUS3 accounts for more than 60% of the total SUS transcripts. One gene, LjSUS2, produces two transcripts due to alternative splicing, a feature not observed in other species to date. We have isolated plants carrying ethyl methanesulfonate-induced mutations in several SUS genes by targeting-induced local lesions in genomes reverse genetics and examined the effect of null alleles of two genes, LjSUS1 and LjSUS3, on nodule function. No differences were observed between the mutants and wild-type plants under glasshouse conditions, but there was evidence for a nitrogen-starvation phenotype in the sus3-1 mutant and severe impairment of growth in the sus1-1/sus3-1 double mutant under specific environmental conditions. Nodules of sus3-1 mutant plants retained a capacity for nitrogen fixation under all conditions. Thus, nitrogen fixation can occur in L. japonicus nodules even in the absence of LjSUS3 (the major nodule-induced isoform of SUS), so LjSUS1 must also contribute to the maintenance of nitrogen assimilation.

TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements

We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus Access to the Lotus TILLING facility can be obtained through http://www.lotusjaponicus.org or http://revgenuk.jic.ac.uk)

A cytosolic invertase is required for normal growth and cell development in the model legume, Lotus japonicus

Neutral/alkaline invertases are a subgroup, confined to plants and cyanobacteria, of a diverse family of enzymes. A family of seven closely-related genes, LjINV1–LjINV7, is described here and their expression in the model legume, Lotus japonicus, is examined. LjINV1 previously identified as encoding a nodule-enhanced isoform is the predominant isoform present in all parts of the plant. Mutants for two isoforms, LjINV1 and LjINV2, were isolated using TILLING. A premature stop codon allele of LjINV2 had no effect on enzyme activity nor did it show a visible phenotype. For LjINV1, premature stop codon and missense mutations were obtained and the phenotype of the mutants examined. Recovery of homozygous mutants was problematic, but their phenotype showed a severe reduction in growth of the root and the shoot, a change in cellular development, and impaired flowering. The cellular organization of both roots and leaves was altered; leaves were smaller and thicker with extra layers of cells and roots showed an extended and broader zone of cell division. Moreover, anthers contained no pollen. Both heterozygotes and homozygous mutants showed decreased amounts of enzyme activity in nodules and shoot tips. Shoot tips also contained up to a 9-fold increased level of sucrose. However, mutants were capable of forming functional root nodules. LjINV1 is therefore crucial to whole plant development, but is clearly not essential for nodule formation or function.

Multiple isoforms of Pisum trypsin inhibitors result from modification of two primary gene products

Characterization of Pisum (pea) seed trypsin inhibitors (TI) and their corresponding cDNAs indicates that the pea TI gene family contains two genes. The existence of multiple TI isoforms can be attributed to post‐translational modifications of primary gene products. Post‐translational processing at the C‐terminus during the desiccation stage of seed development results in the appearance of TI isoforms with increased affinity for the target enzyme, trypsin.

A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

Transcription Factors of Lotus: Regulation of Isoflavonoid Biosynthesis Requires Coordinated Changes in Transcription Factor Activity

Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors.

Combinatorial variation in coding and promoter sequences of genes at the Tri locus in Pisum sativum accounts for variation in trypsin inhibitor activity in seeds

Abstract. Cultivars of Pisum sativum that differ with respect to the quantitative expression of trypsin/chymotrypsin inhibitor proteins in seeds have been examined in terms of the structure of the corresponding genes. The patterns of divergence in the promoter and coding sequences are described, and the divergence among these exploited for the development of facile DNA-based assays to distinguish genotypes. Quantitative effects on gene expression may be attributed to the overall gene complement and to particular promoter/coding sequence combinations, as well as to the existence of distinct active-site variants that ultimately influence protein activity. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00438-002-0667-4.

Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement

The promoter from one of the two seed-expressed genes encoding trypsin/chymotrypsin inhibitors (TI) has been isolated and characterised in transgenic pea lines, following its re-introduction by Agrobacterium-mediated transformation, as a TI promoter-beta-glucuronidase (GUS) gene fusion. The promoter from this gene (TI1) directed expression of GUS enzyme at late stages of embryogenesis, comparable to those determined for activity of the homologous native TI genes. GUS expression was detected in roots of plants subjected to drought stress conditions, indicating that the TI1 gene, normally seed-specific in its expression, can be induced under these conditions. A second gene construct utilised the TI1 gene promoter to direct expression of an antisense TI gene. Seed TI activities in some lines transformed with this construct were reduced significantly. A limitation of the pea transformation methodology for antisense manipulations, in particular, is the observed frequency of non-transmission of transgenes from primary transformants (up to 80%).

Expression patterns of genes encoding seed trypsin inhibitors in Pisum sativum

In Pisum sativum (pea) seeds, trypsin inhibitor (TI) activity increased in both axes and cotyledons during seed development. TI activities were appreciably higher in axes than in cotyledons and gel analyses showed that some TI isoforms accumulated differentially in the two organs. The maximal amounts of TI mRNA were very similar in axes and cotyledons but, in contrast to increased TI activities, RNA amounts decreased during desiccation in both organs; however, there were differences between the two organs in the temporal pattern of mRNA accumulation. In situ hybridization analyses of developing axes showed the presence of TI mRNA initially in the root cap, at later stages in parenchyma cells of the radicle and finally in the shoot apex. Immunolocalization of TI protein in developing axes showed that its spatial distribution was very similar to that of the mRNA. The organ specificity of TI gene expression was investigated by Northern analyses, which showed that mRNA corresponding to seed-expressed TI genes was not present in vegetative organs of the plant under normal growing conditions. TI mRNA was induced, however, in roots of plants that had been subjected to drought conditions, suggesting a possible function for these proteins in dehydrating plant tissues.