We have searched for induced transcripts in a cDNA library derived from bean cell supension cultures treated with an elicitor from Colletotrichum lindemuthianum. Six independently isolated cDNAs corresponding to rapidly induced small mRNAs have been classified by their DNA sequence and slightly different induction behaviour into two groups. 5'- and 3'-untranslated regions exhibit little similarity, but the deduced small acidic proteins designated PvPR1 and PvPR2 are 89% identical. No relationship was found with the well-characterized PR1 proteins from tobacco. However, the PvPR proteins are closely related to pI49 in pea (64% identity), pSTH2 in potato (41% identity) and PcPR1-1 in parsley (39% identity), which are also induced in response to elicitor or microbial attack. Moreover, a major pollen allergen in birch (BetvI) has a 44% identity with PvPR1 proteins. These similarities establish a ubiquitous class of conserved defense-related proteins and suggest a common yet still unknown function. Southern blot analysis indicates that PvPR protein gene organization is highly complex with an estimated copy number of more than 12 genes.

The intracellular pathogenesis-related (PR) proteins of common bean (Phaseolus vulgaris L.) are encoded by a highly polymorphic family of at least 20 genes. One member, the Ypr10*c gene, has been isolated and characterised. The deduced amino acid sequence of the encoded protein, PR-10, exhibits similarities to tree-pollen allergens, to food allergens from celery and apple and to ginseng ribonuclease peptide sequences. We show by RNA blot analysis that the Ypr10 gene family, including Ypr10*c, is strongly expressed in bean roots. In leaves Ypr10 transcript levels are low in young and mature stages but are elevated during senescence and in diseased states. Dark treatment of leaves causes strong induction of Ypr10 transcripts, which is reversible by light, and diurnal rhythms of transcript accumulation during the night are observed. Ypr10 genes are responsive to external stimuli related to pathogen-defence such as glutathione or salicylic acid. Transcriptional activity of a Ypr10*c promoter-beta-glucuronidase fusion gene in transgenic tobacco was observed in roots, in developing xylem and phloem of stems, and in the blade of senescent leaves, with highest levels at the onset of senescence. The most striking characteristic of developmental expression was the specific localisation of beta-glucuronidase activity in the transmitting tract of styles in flowers at anthesis. Feeding of various pathogen-related and stress-related stimuli to young tobacco leaves led to accumulation of GUS activity in leaf blades. We identify considerable spatio-temporal similarities between reported expression patterns of Ypr10 genes and ribonuclease genes, which, together with the significant sequence similarity to the ginseng ribonuclease, support the hypothesis of a ribonuclease function for PR-10 proteins and allow the prediction of possible biological roles.

NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) catalyzes the oxidative decarboxylation of malate to pyruvate, producing CO, and NADPH. We have examined regulatory properties of a 2.8-kb promoter-leader fragment of a bean (Pbaseolus vurgaris 1.) NADP-ME gene (PvME1) predicted to encode a cytosolic form of the enzyme by expression analysis of promoter-P-glucuronidase fusions in transgenic tobacco plants. l h e PvMEl promoter directed strong expression in stems, which was confined to vascular and pith tissues, and was also active in floral and reproductive tissues. Wounding caused a marked induction of promoter activity, which was further strongly enhanced upon application of stimuli related to pathogen defense. Clutathione (reduced form) was the strongest inducer, but oxidized glutathione, funga1 elicitor, cellulase, catalase, ascorbic acid, and NADPH were additional potent promoterstimulating agents. Responsiveness to reduced glutathione was also shown at the leve1 of PvMEl mRNA accumulation in bean plants. l h e putative contributions of NADP-ME gene expression to the plant defense response and possible mechanisms of defense gene regulation by conditions of oxidative stress as well as by H,O, and antioxidant levels are discussed.

Azosprilla were collected in wheat fields from subtropical and temperate soils of central Nepal at various elevations. Different wheat cultivars responded positively and significantly in grain yield, grain N-yield, and total N-yield in plant shoots to the inoculation with Nepalese isolate Azospirillum 10SW. Nepalese wheat cv. Seto responded significantly better with Azospirillum 10SW than with the Brasilian isolate A. lipoferum Sp 108 st, a strain which was found highly efficient in earlier experiments with German wheat cultiyars, especially cv. Turbo. Yield of Turbo was increased by inoculations of both Azospirillum strains too, but it showed no significant differences depending from the inoculum used. The higher efficacy of combining Azospirillum 10SW and Seto, both collected from the same locality, indicates the possibility of improved associations using traditional cultivars and local bacteria.

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Media from the in vitro association of wheat and Azospirillum lipoferum and from wheat plants alone proved to be chemotactically active. Medium from wheat plants showed a higher attraction than medium from the association. The main attractants were sucrose, glucose, and fructose. In mineral medium without any added sugars, and in association medium with sucrose supplied, and from wheat roots alone, a sucrose excretion and an active invertase were detected. By cleaving sucrose the chemotactic potential increased. Sucrose can not be metabolized by A. lipoferum, whereas glucose and fructose are. Utilization of glucose and fructose by the bacteria may explain why medium from the association wheat–Azospirillum was less chemotacticaly active than medium from wheat plants alone. Cleavage of sucrose has the additional effect of providing energy sources for bacterial growth and dinitrogen fixation.

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Dialkyl azodiformates form dihydrooxadiazines with indene, dihydro-1,4-dioxine, vinylene carbonate, cisand irons-1,2-dimethoxyethylene, and vinyl acetate by 1,4 addition; 1,2 addition yielding diazetidines is observed with vinyl ethers. Diazetidines also result from the addition of 4-phenyl-A'-1,2,4-triazoline-3,5-dione (PTD) to indene and dihydro-1,4-dioxine. Dihydrooxadiazines are formed in a concerted Diels-Alder reaction with inverse electron demand, the diazetidines via dipolar intermediates. The acceleration of azodiformate addition by illumination is due to the photochemical production of as azodiformates, which show increased thermal reaction rates compared with the irons isomers.