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The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3kcal/mol to set the enzyme into a catalytically competent form.

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in Δ C p , with NADP binding Δ C p closer to what is expected of crystallographic structures, while NADPH Δ C p is three times larger. The estimated dimer–tetramer association constant is 1.5 · 10 6 M − 1 , and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.

summary The gene rolB from the T-DNA of Agrobacterium rhizogenes is capable of directing differentiation of roots in transformed plant cells. A detailed histological analysis of the expression of this gene in transgenic tobacco plants is presented here. Gene fusions between the promoter of rolB and the GUS reporter gene (encoding E. coliβ-glucuronidase) were utilized: tissue and cell specific GUS activity as driven by a long (1185 base pairs) and a short (306 bp) version of the rolB promoter was visualized histochemically during different phases of plant development, from embryo to flowering. The extended (1185bp) rolB promoter is under developmental control and is specifically active in the initial cells of all types of meristems and in the vascular system (phloem, phloem parenchyma, xylem parenchyma, pericycle and pericycle-like cells) of mature organs as well as in the central cylinder of the embryo. In contrast, the 306 bp deletion of the promoter is unable to drive expression in menstematic initials but is still active in the vascular system of the seedling.

In Phaseolus vulgaris (bean), PGIP is encoded by a gene family. We have isolated cDNA clones corresponding to two different pgip genes (pgip-1 and pgip-2). These genes have been separately expressed in Nicotiana benthamiana using the potato virus X (PVX) as a vector. PGIP-1 and PGIP-2 in crude protein extracts of PVX-infected TV. benthamiana, and a PGIP-1 purified from transgenic tomato plants carrying the pgip-1 coding sequence under the control of the CaMV 35S promoter, have been assayed for specificity of interaction with several fungal PGs. PGIP-1 from both plant sources exhibited a similar specificity, which was different from that of PGIP-2 and of the bulk PGIP purified from bean. Notably, PGIP-1 was unable to interact with homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while PGIP-2 and the bulk bean PGIP interacted with this enzyme.

The expression and the effects of Agrobacterium rhizogenes rolB gene during in vitro flower, root and vegetative organogenesis were investigated. Thin cell layers were excised from floral pedicels, inflorescence rachis and stems of tobacco plants containing either rolB and its promoter fused to the GUS reporter gene or only this latter construct. Organogenesis and GUS activity were monitored in pedicel and stem expiants cultured under conditions for floral, root, and vegetative neoformation. Flowering, rhizogenesis and caulogenesis were enhanced in rolB-transformed expiants as compared to untransformed controls: the neoformed meristems were much more numerous and appeared sooner. rolB promoter was specifically active in the initial cells of all the meristem types. Rachis expiants from rolB-plants produced, upon culture under flowering conditions, also an exceeding number of vegetative buds. Thus, rolB seems to stimulate the formation of meristems regardless of their subsequent differentiation and of the hormone controlling the morphogenic event.

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5' non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (-623 to -471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (-341 to -306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C + D + E, B + C + D, B + C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B + C + E is active only in vascular meristematic cells. Domain C (-216 to -158) seems to have a double regulatory role as construct B + E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.

The rol genes are part of the T-DNA which is transferred by Agrobacterium rhizogenes in plant cells, causing neoplastic growth and differentiation. Each of these bacterial oncogenes deeply influences plant development and is finely regulated once transferred into the plant host. Both from the study of the effects and biochemical function of the rol genes and from the analysis of their regulation, important insight in plant development can be derived. Some of the most intriguing aspects of past, current and future research on this gene system are highlighted and discussed.

summary The expression and effects of the plant oncogene rolB during in vitro flower and root morphogenesis were investigated. Thin cell layers (TCL) were excised from floral pedicels and from stems of transgenic tobacco plants containing either rolB and the promoter of rolB fused to the GUS reporter gene (BpB–GUS plants) or only this latter construct (pB-GUS plants). Organogenesis and GUS activity were monitored in pedicel and stem TCLs for 30 days under conditions of floral or root neoformation. Both flowering and rhizogenesis are substantially enhanced and more precocious in rolB-transformed explants than in TCLs from pB-GUS and control plants. Most interestingly, both in flower arid in root neoformation, meristemoids are much more numerous and appear sooner in rolB-transformed tissues. Thus, the effect of rolB does not seem to be specifically the induction of rooting, but, rather, the promotion of meristem formation, regardless of subsequent differentiation. In addition, we provide evidence that the presence of the gene product of rolB does not alter the pattern and timing of expression of the gene.

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5' non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions -623 to -471 and from -471 to -341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position -1185 to -341, although at different levels, maintained the same pattern of GUS expression-maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from -341 to -306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.

Agrobacterium rhizogenes is responsible for the hairy root syndrome of dicotyledonous plants which consists in an abundant proliferation of roots at the wounded site of bacterial infection (Elliot, 1951). Hairy roots contain a portion (T-DNA) of a large bacterial plasmid (Ri plasmid), which directs growth and differentiation of the transformed plant cells (Chilton et al., 1982; Spano et al-, 1982b; White et al., 1982; Willmitzer et al.,1982). Hairy roots grow very actively in vitro in the absence of hormones with a characteristic highly branched and plagiotropic pattern (David et al., 1984); they synthesize specific opines of which three major types have been so far identified, agropine, mannopine and cucumopine (Petit and Tempe., 1985), corresponding to different families of Ri plasmids (Costantino et al., 1981). Whole, fertile plants of different species have been regenerated from hairy root tissues in several laboratories (Chilton et al., 1982; Spano et al., 1982a; Tepfer, 1984; Spano et al., 1987).

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA + B + C) and by rolB alone provided an extended segment beyond its 5' non-coding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB + C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13 + 14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13 + 14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA + B + C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA + B + C roots by the concomitant presence of ORF13 + 14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.

Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the TR-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (∼1200 bp) at the 5′ end of rolB had to be included in the construction. A shorter (∼300 bp) 5′ region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5′ regions of rolB were then cloned upstream of the β-glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.

Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.