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.

Root starch accumulation in response to arbuscular mycorrhizal colonization differs among Lotus japonicus starch mutants.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts dependent for completion of their life cycle on plant carbohydrates, which they trade for mineral nutrients. Plant colonization by AM fungi is therefore expected to induce profound changes in plant carbon metabolism. We have previously observed that on one hand starch accumulation increases in responses to pre-symbiotic fungal signals and on the other hand, it decreases in mycorrhizal Lotus japonicus roots (Gutjahr et al. in New Phytol 183:53-61, 2009). To examine the importance of starch metabolism for AM development, we took advantage of a novel series of Lotus japonicus mutants impaired either in starch degradation or in synthesis. Normal AM colonization in all mutants indicated that defects in starch metabolism do not affect AM development and that carbohydrates can be supplied to the AM fungus without a requirement for starch synthesis. Furthermore, our experiments allowed us to characterize root starch dynamics in detail and point to continued turnover of starch in the degradation mutants in the presence of mycorrhiza.

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.

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.

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.

Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants.

Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression patterns, were isolated and characterized from Pisum. Under standard plant growth conditions, one class is expressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall sequence, active site sequences and inhibitor function.