We have characterized the development of seeds in the model legume Lotus japonicus. Like soybean (Glycine max) and pea (Pisum sativum), Lotus develops straight seed pods and each pod contains approximately 20 seeds that reach maturity within 40 days. Histological sections show the characteristic three developmental phases of legume seeds and the presence of embryo, endosperm, and seed coat in desiccated seeds. Furthermore, protein, oil, starch, phytic acid, and ash contents were determined, and this indicates that the composition of mature Lotus seed is more similar to soybean than to pea. In a first attempt to determine the seed proteome, both a two-dimensional polyacrylamide gel electrophoresis approach and a gel-based liquid chromatography-mass spectrometry approach were used. Globulins were analyzed by two-dimensional polyacrylamide gel electrophoresis, and five legumins, LLP1 to LLP5, and two convicilins, LCP1 and LCP2, were identified by matrix-assisted laser desorption ionization quadrupole/time-of-flight mass spectrometry. For two distinct developmental phases, seed filling and desiccation, a gel-based liquid chromatography-mass spectrometry approach was used, and 665 and 181 unique proteins corresponding to gene accession numbers were identified for the two phases, respectively. All of the proteome data, including the experimental data and mass spectrometry spectra peaks, were collected in a database that is available to the scientific community via a Web interface (http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi). This database establishes the basis for relating physiology, biochemistry, and regulation of seed development in Lotus. Together with a new Web interface (http:// bioinfoserver.rsbs.anu.edu.au/utils/PathExpress4legumes/) collecting all protein identifications for Lotus, Medicago, and soybean seed proteomes, this database is a valuable resource for comparative seed proteomics and pathway analysis within and beyond the legume family.

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyureainduced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.

Legume pods serve important functions during seed development and are themselves sources of food and feed. Compared to seeds, the metabolism and development of pods are not well-defined. The present characterization of pods from the model legume Lotus japonicus, together with the detailed analyses of the pod and seed proteomes in five developmental stages, paves the way for comparative pathway analysis and provides new metabolic information. Proteins were analyzed by two-dimensional gel electrophoresis and tandem-mass spectrometry. These analyses lead to the identification of 604 pod proteins and 965 seed proteins, including 263 proteins distinguishing the pod. The complete data set is publicly available at http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi , where spots in a reference map are linked to experimental data, such as matched peptides, quantification values, and gene accessions. Identified pod proteins represented enzymes from 85 different metabolic pathways, including storage globulins and a late embryogenesis abundant protein. In contrast to seed maturation, pod maturation was associated with decreasing total protein content, especially proteins involved in protein biosynthesis and photosynthesis. Proteins detected only in pods included three enzymes participating in the urea cycle and four in nitrogen and amino group metabolism, highlighting the importance of nitrogen metabolism during pod development. Additionally, five legume seed proteins previously unassigned in the glutamate metabolism pathway were identified.