A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development.

Abstract

To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 @. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganlsms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development. INTRODUCTIONActinorhizal root nodules are induced by actinomycetes of the genus Frankia on severa1 woody dicotyledonous plant species belonging to eight different plant families (Benson and Silvester, 1993). The tissue organization of single lobes of these nodules resembles that of lateral roots in that they contain a central vascular bundle. However, they lack root caps and have both infected and uninfected cortical cells (Berry and Sunell, 1990). The formation of these lobes is initiated in the pericycle. The structure of actinorhizal nodules is dissimilar to nodules induced by Azorhizobium, Bradyrhizobium, or Rhizobium on legume roots (Hirsch, 1992), which are initiated in the root cortex of the plant and contain peripheral vascular bundles, yet similar to that of nodules induced by Bradyrhizobium or Rhizobium on Parasponia, the only non-legume nodulated by Rhizobium (Trinick, 1979).Similar to nodules formed on temperate legumes, actinorhizal nodule lobes have an indeterminate growth pattern due to the presence of an apical meristem that differentiates continuously in a proximal direction (Berry and Sunell, 1990; Hirsch, 1992). A part of the new cortical cells formed by the meristem is subsequently infected by bacterial hyphae. This continuous production and infection of new cells leads to a 1 To whom correspondence should be addressed. zonation of the nodule lobe. Thus, starting from the apical meristem, we can distinguish four zones. The meristematic zone (zone 1) consists of small dividing cells that do not contain bacteria. The prefixation zone (zone 2) contains enlarging cortical cells. Some of them are infected and in turn enlarge more than uninfected cells, while being gradually filled with hyphae from the center outward (Lalonde, 1979;Schwintzer et al., 1982;Berry and Sunell, 1990). When...

The seed coat-specific expression of a subtilisin-like gene, SCS1 , from soybean

“…In contrast to the bacterial BPN, yeast Kex2 or mammalian furin subtilisins, which share 7±24% sequence similarity with SCS1, SCS1 has a sequence insertion between the conserved asparagine and serine residues. This appears to be a feature unique to plant subtilases (Yamagata et al 1994;Ribeiro et al 1995;Tornero et al 1997). The sequence alignments predict that SCS1 has a signal sequence of 23 residues followed by a prosequence of 93 residues and a mature peptide domain of 654 residues (Fig.…”

“…They coded for AF70 from Picea abies (Sabala et al 1997), cucumisin from Cucumis melo (Yamagata et al 1994), P69B from Lycopersicon esculentum (Tornero et al 1997), Ag12 from Alnus glutinosa (Ribeiro et al 1995). These proteins belong to the Pyrolysin family in the clan of serine proteases known as the subtilases and share 32±47% sequence similarity with SCS1.…”

“…In addition to soybean SCS1, they include Arabidopsis AIR3 (Neuteboom et al 1999), black spruce AF70 (Sabala et al 1997), musk melon cucumisin (Yamagata et al 1994), tomato P69B (Tornero et al 1997;Vera et al 1989), lilly LIM3 (Taylor et al 1997), black alder Ag12 (Ribeiro et al 1995) and tomato SBP50 (Schaller and Ryan 1994). Sequence analysis predicts that they all have signal peptides and the properties of secreted proteins.…”

“…For instance, P69B and Ag12 have been implicated respectively in plant defense and alderFrankia symbiotic interactions (Tornero et al 1997;Ribeiro et al 1995). It is interesting that pathogens such as Verticillium dahliae also secrete a trypsin-like serine protease to the extracellular matrix during dierentiation (Dobinson et al 1997).…”

SEP-1 - a subtilisin-like serine endopeptidase from germinated seeds of Hordeum vulgare L. cv. Morex

“…Nevertheless, the optimum pH for its activity was lower than those of most similar enzymes, whose optimal values are around pH 9.0 (Kaneda and Tominaga 1977;Vera and Cornejero 1988;Curroto et al 1989;Rudenskaya et al 1998) or higher (Uchikoba et al 1990). Like that of SEP-1, the molecular weights of the cucumisin-like enzymes from Cucurbitaceae and from other plant families, are generally around 70 kDa (Vera and Cornejero 1988;Yamagata et al 1994;Ribeiro et al 1995;Rudenskaya et al 1995Rudenskaya et al , 1998Sabala et al 1997).…”