The synthesis of Cd-binding peptides (CdBPs) was induced upon addition of 20 micromolar CdCI2 (nonphytotoxic level) to the nutrient solution of hydroponically grown tobacco seedlings (Nicotiana rustica var Pavonii). Amino acid analysis showed that the main components were -y-(GIu-Cys)3-Gly and -y-(GIu-Cys)4-Gly.Seedlings exposed to the metal for 1 week contained similar glutathione levels as found in the controls (about 0.18 micromole per gram fresh weight). If, as has been proposed, CdBPs are involved in Cd-detoxification by chelation, both metal and ligand must be localized in the same cellular compartment. To directly determine the localization of Cd and CdBPs, protoplasts and vacuoles were isolated from leaves of Cd-exposed seedlings. Purified vacuoles contained virtually all of the CdBPs and Cd found in protoplasts (104% ± 8 and 110% ± 8, respectively). CdBPs were associated with the vacuolar sap and not with the tonoplast membrane. Glutathione was observed in leaves and protoplasts but not in vacuoles. The probability that CdBPs are synthesized extravacuolarly and our finding that they and Cd are predominantly located in the vacuole suggest that these molecules might be involved in transport of Cd to the vacuole. Our results also suggest that a simple cytoplasmic chelator role for CdBPs in Cd tolerance cannot be assumed.Various mechanisms have been proposed for detoxification of potentially toxic heavy metal ions in plants. Metal ions such as Cd (also Hg, Pb, and Cu) can interfere with the plant's metabolism by binding to essential sulfhydryl groups of enzymes or structural proteins. Therefore, it seems necessary to keep the intracellular concentration of potentially toxic heavy metal ions at a low level. This can be achieved by a number ofdifferent mechanisms (31). In Cd-resistant Euglena gracilis, for instance, decreased metal uptake has been proposed as the principal mechanism of metal tolerance (1). Extracellular sequestration ofCd and active metal efflux have been reported for some bacteria (18, 32 (14). Particularly in the case ofCd toxicity, however, most attention has centered on cysteine-rich y-glutamyl peptides called CdBPs or phytochelatins which are highly induced by Cd and bind it with high affinity (8,21,25,28). In contrast to metallothioneins, which are considered to be a principal means for metal detoxification in animal systems and some fungi (31), CdBPs are not primary gene products (24). They are structurally related to GSH and their biosynthetic pathway probably involves GSH or metabolites of GSH.Several authors (8,22,25,28) have reported positive correlations between the occurrence of CdBPs and tolerance to Cd. If chelation of Cd by CdBPs in the cytosol is a principal mechanism of tolerance, then the bulk of both metal and ligand must be localized in this compartment. Current evidence concerning the subcellular localization of Cd is not entirely consistent. Rauser and Ackerley (20) found Cd associated with electron dense granules in the cytoplasm, vacuoles and nuclei of Agros...

Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum 1. cv Havana 425 seeds. Treatment with 1 O-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of P-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I P-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of /3-1,3-glucanase accumulation but did not delay the onset of induction. lndependent of the ABA concentration used, onset of endosperm rupture was correlated with the same P-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I p-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.

Legumes form tripartite symbiotic associations with nodule-inducing soil bacteria of the genera Rhizobium, Brudyrhizobium, or Azorkizobium (Caetano-Anollés and Gresshoff, 1991;Hirsch, 1992) and with VAM fungi (BonfanteFasolo, 1987;Koide and Schreiner, 1992). Both the rhizobial and fungal microsymbionts improve the mineral nutrition of the host plant in exchange for assimilates provided by the latter. The nitrogenase enzyme of rhizobia fixes atmospheric nitrogen in the nodules (Thorneley, 1992), and fungal hyphae facilitate the uptake of ions, mainly phosphate, in mycorrhizal roots (Smith and Gianinazzi-Pearson, 1988). In most cases investigated, especially when both nitrogen and phosphate are limiting factors, VAM fungi '

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