Construction of transgenic pea lines with modified expression of diamine oxidase and modified nodulation responses with exogenous putrescine.

Diamine oxidase (DAO) might influence pea nodule development either by regulating the peroxide-driven cross-linking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity in extracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing co-suppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in the cross-linking of matrix glycoprotein, indicating that putrescine-derived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serve to reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues.

Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: implications for pea nodule initiation by Rhizobium leguminosarum.

Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.

Isolation of lipoxygenase cDNA clones from pea nodule mRNA.

The pattern of lipoxygenase (LOX) gene expression was investigated in pea nodule tissues using the technique of in situ hybridization. Five lipoxygenase cDNAs were cloned from nodule mRNA by the RT-PCR and 3' RACE procedures. These clones (loxN1 to loxN5) show a high degree of sequence homology, except in the 3'-untranslated region. Gene-specific riboprobes were therefore generated from subclones carrying the 3'-untranslated regions in order to investigate tissue-specific gene expression. Northern blotting analysis revealed that loxN1 corresponded to a transcript that was expressed exclusively in roots and nodules but not in the aerial parts of the plant. However, none of the LOX genes appeared to be up-regulated in nodule tissue relative to uninfected roots. Starting with the incomplete cDNA clone for loxN1, the full coding sequence termed lox1:P.s:1 was obtained by further rounds of RT-PCR and 5' RACE procedures. In situ hybridization with nodule tissues revealed several different patterns of expression for the various LOX probes. However, none of the corresponding transcripts was expressed exclusively in the invasion zone, as might have been expected if one LOX gene product had been uniquely associated with the invasion process. In conclusion, this study provides no evidence for a direct role for any LOX gene product in plant-microbe interaction or host defence, but the fact that all the transcripts were expressed at the nodule apex suggests that LOX could be involved in the development of this organ.

The extensin multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures.

Changes in extensin gene expression were examined in cultured tomato cells following treatments leading to the production of activated oxygen species. Digitonin, a steroid glycoalkaloid compound, has been shown to trigger a rapid and transient production of superoxide anion, O2-*. 6 h after application of 50 or 100 microM of digitonin, the accumulation of four extensin transcripts (1.5, 2.6, 4.0 and 6.1 kb) was observed. Superoxide dismutase strongly inhibited the digitonin-mediated response, suggesting a key role of O2-* in the signalling cascade. Furthermore, cells treated with enzymatically produced O2-* generated by xanthine oxidase (0.015 U/ml) gave a similar extensin response and again, SOD exerted a strong inhibitory effect on the response. On the other hand, H2O2 (2 mM) or the enzymatic H2O2 generator, glucose oxidase (0.34 U/ml), elicited the accumulation of only three of the four transcripts (1.5, 2.6 and 4.0 kb), indicating that the corresponding genes could be regulated either by H2O2 or O2-* but that the gene encoding the 6.1 kb transcript was exclusively expressed in response to O2-*. Finally, it was shown that lipid peroxidation, which was only induced when cells were exposed to H2O2, did not participate in the AOS-mediated gene expression for extensin. It can be concluded from these results that tomato cells are able to discriminate H2O2 from O2-* and they probably sense the latter by the specific oxidation of an extracellular component.

Modulation of carbohydrate-binding capacities and attachment ability of Bradyrhizobium sp. (lupinus) to white lupin roots.

The attachment of Bradyrhizobium sp. (Lupinus) strain MSDJ718 to excised roots of white lupin was examined. Maximal attachment occurred at early to middle log phases of bacterial growth. This binding was pH dependent, with an optimal value reached at 6.6. Irrespective of the culture age, the attachment was strongly affected by the calcium concentration of the growth medium: a Ca2+ limitation in the Bergersen medium led to optimal attachment of the bacteria. When L-fucose was added during the attachment assay, a significant inhibition was observed. The binding was stimulated when bacteria were cultivated with lupin root extracts, genistein and genistin (two lupin isoflavonoids), and some monosaccharides. In addition, with a spectrofluorimetric method using fluoresceinylated neoglycoproteins, it was shown that the increase of the attachment of bacteria to host cells was correlated to the increase of the L-fucoside binding capacity of the rhizobial cells. Taken together, the results obtained in the present study evidenced a possible role of the L-fucose specific bacterial lectin previously described in the Rhizobium-lupin host cell recognition.

Purification of an alpha-L-fucoside-binding protein from Rhizobium lupini.

Lectins associated with the bacterial cell surface of Rhizobium lupini strain LL13 were evidenced by erythrocyte agglutination, by aggregation of neoglycoprotein coated beads and by spectrofluorimetry using fluoresceinylated neoglycoproteins. At pH 5.0, a specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucose was observed. The binding of this labelled neoglycoprotein is a saturable phenomenon and is inhibited by the same unlabelled neoglycoprotein. Extracts of R lupini obtained by disrupting a bacterial pellet through a French press were stabilized at pH 5.6 by gel filtration and purified to homogeneity by affinity chromatography on Agarose A4 substituted with alpha-L-fucose. A protein with a M(r) approximately 19,000 was specifically eluted from this affinity column with L-fucose. Isoelectric focusing of this sample yielded a single band with pI near 6.7. This protein specifically aggregated L-Fuc-BSA-coated microspheres. The results obtained in the present study indicate that we have purified from Rhizobium lupini strain LL13, a L-fucose binding protein as a lectin.