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ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5 end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules.translation ͉ short open reading frames ͉ peptide signals T he nodule on the roots of legumes is an organ induced by rhizobia in which they are hosted intracellularly and where they find an ideal environment for symbiotic nitrogen fixation. The plant genes that are specifically induced by nodulation factor-secreting rhizobia during early stages of nodule development have been termed early nodulin (ENOD) genes. Among these genes, ENOD40 is one of the earliest nodulins and appears to play an important role in root nodule organogenesis. ENOD40 is induced by nodulation factors and the phytohormone cytokinin, and its expression precedes the first cortical cell division (1, 2). In mature nodules, the expression of ENOD40 has been detected in cells surrounding vascular bundles (3, 4). In addition, this gene also is expressed at low levels in stem and root cells (3). Recent work (5) has revealed ENOD40 homologs also in the monocotyledonous plants rice and maize. ENOD40 is therefore widespread in the plant kingdom, suggesting that it may have a general biological function.A remarkable feature of legume ENOD40 genes is that they contain only short ORFs. Therefore, it was initially proposed that this gene functions as an RNA (6, 7). All ENOD40 genes studied thus far contain two highly conserved regions. Recently, it was reported that the 5Ј located conserved region I of soybean (Glycine max) ENOD40 encodes a small peptide (8). This work claimed that the peptide renders tobacco cells insensitive to high concentrations of auxin. However, these data were obtained by counting tobacco cells undergoing division and could not be reproduced by using other proliferation assays (9). Moreover, a study with transgenic clover containing an auxin-responsive promoter-␤-glucuronidase (GUS) fusion failed to show the involvement of this peptide in perturbing auxin balance (10). Although genetic approaches using translational fusions were used to study ENOD40 expression (11,12), no direct biochemica...

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum . Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration-and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.

Four independent recombinant lambda clones hybridizing to parsley chalcone synthase (CHS) cDNA were isolated from a soybean (Glycine max) genomic library. Restriction fragment length polymorphism (RFLP) analysis indicated that the CHS gene family comprises six members. The CHS genes were found to be clustered with three genes on a 10 kb segment and pairs on others. DNA sequences of the 5'-, the coding-, and the 3' untranslated regions were determined for three different genes. A consensus alignment of the 5' regions revealed extensive homology between them for up to 150 bp upstream of the TATA box. Developmental regulation of CHS was observed in uninfected and in rhizobium-infected roots. Regulation at the level of transcription by different stimuli was investigated in the root, stem and cotyledons of soybean seedlings. Our results suggest a co-operative induction of CHS genes by wounding and elicitor treatment of cotyledons. The most rapid transcript accumulation, however, was observed in roots and stems. The induction of CHS genes by light was found to be UV dependent. A possible involvement of different members of the CHS gene family in response to elicitor versus UV treatment was analysed by the use of gene specific probes, and unexpectedly revealed that only CHS 1 transcription was induced by either elicitor or UV treatment of seedlings.