From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement.

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

Global gene expression profiling during Medicago truncatula-Phymatotrichopsis omnivora interaction reveals a role for jasmonic acid, ethylene, and the flavonoid pathway in disease development.

Phymatotrichopsis omnivora (Duggar) Hennebert causes a destructive root rot in cotton, alfalfa (Medicago sativa), and many other dicot species. No consistently effective control measures or resistant host germplasm for Phymatotrichum root rot (PRR) are known. The relative genetic intractability of cotton and alfalfa precludes their use as model pathosystem hosts for P. omnivora. Therefore, we used the model legume M. truncatula and its available genetic and genomic resources to investigate PRR. Confocal imaging of P. omnivora interactions with M. truncatula roots revealed that the mycelia do not form any specialized structures for penetration and mainly colonize cortical cells and, eventually, form a mycelial mantle covering the root's surfaces. Expression profiling of M. truncatula roots infected by P. omnivora identified several upregulated genes, including the pathogenesis-related class I and class IV chitinases and genes involved in reactive oxygen species generation and phytohormone (jasmonic acid and ethylene) signaling. Genes involved in flavonoid biosynthesis were induced (2.5- to 10-fold over mock-inoculated controls) at 3 days postinoculation (dpi) in response to fungal penetration. However, the expression levels of flavonoid biosynthesis genes returned to the basal levels with the progress of the disease at 5 dpi. These transcriptome results, confirmed by real-time quantitative polymerase chain reaction analyses, showed that P. omnivora apparently evades induced host defenses and may downregulate phytochemical defenses at later stages of infection to favor pathogenesis.

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots.

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H2O2 accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl3, intracellular accumulation of H2O2 was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H2O2 was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.