Irma Vijn

A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter.

The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions −95 and −77 in the PsENOD12B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way. ENBP1 is expressed in the same cell types as ENOD12. However, additional expression is observed in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5′-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.

Fructosyltransferase mutants specify a function for the beta-fructosidase motif of the sucrose-binding box in specifying the fructan type synthesized

The onion fructosyltransferase fructan:fructan 6G-fructosyltransferase (6G-FFT) synthesizes fructans of the inulin neo-series using 1-kestose as a substrate. 6G-FFT couples a fructosyl residue to either the terminal glucose via a β(2-6) linkage or a terminal fructose via a β(2-1) linkage. The sucrose-binding box is present at the N-terminus of invertases and fructosyltransferases. We tested its function by producing swaps of the first 36 amino acids of 6G-FFT with that of onion sucrose:sucrose 1-fructosyltransferase (1-SST) (SST-GFT) and vacuolar invertase (INV-GFT). In contrast to 6G-FFT, invertase and 1-SST are able to utilize sucrose as their only substrate. The chimerical enzymes were unable to use sucrose, but were active when incubated with 1-kestose. INV-GFT synthesized a similar array of fructans as 6G-FFT, in contrast, SST-GFT showed a dramatic shift in activity towards synthesis of β(2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the β-fructosidase motif, which is part of the sucrose-binding box, consists of NDPNG/A. This motif is variable in fructosyltransferases and consists of NDPSG in 6G-FFT and ADPNA in 1-SST of onion. We studied the importance of the 6G-FFT β-fructosidase motif using mutants S87N (NDPNG) and N84A;S87N (ADPNG). S87N has 6G-FFT activity, whereas N84A;S87N has a activity that was shifted towards synthesis of β(2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the β-fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.

Expression of fructosyltransferase genes in transgenic plants

Fructans serve as a carbohydrate reserve in many plant species and are also synthesised by several microorganisms. Over the past decade interest in the use of fructans for food and non-food applications has increased exponentially. Our interest is to modify crops for the production of tailor-made fructans. Therefore we introduced genes encoding bacterial fructosyltransferases into several non-fructan storing plants, e.g. tobacco and potato. Different cellular targeting sequences were used for the expression of the bacterial levansucrases in transgenic tobacco and potato plants resulting in varying levels of fructan and often in changes in the phenotype.

The vetch (Vicia) and Rhizobium leguminosarum bv. viciae symbiosis: A system to study the activity of Rhizobium Nod factors.

The interactions of leguminous plants and bacteria of the genera Rhizobium, Bradyrhizobium and Azorhizobium -here collectively called rhizobia- result in the formation of root nodules, new organs in which the bacteria are able to fix atmospheric nitrogen into ammonia. The formation of root nodules involves a number of developmental steps (Newcomb, 1976; Newcomb, 1981). First the bacteria attach to the root hairs of the host plant and cause deformation and curling of root hairs. Then the bacteria enter the plant by newly formed tubular structures, the infection threads, starting from the curls of the root hairs. At the same time, cells in the cortex of the root become mitotically active and form a nodule primordium. The infection threads grow towards the nodule primordia, and after penetration of individual primordium cells the bacteria are released in the cytoplasm, by endocytosis. Then the nodule primordium differentiates into a nodule.

Identification of Trans-Acting Factors Regulating Nodulin Gene Expression

Functional studies of nodulin gene promoters in transgenic legumes have identified a number of cis-regulatory elements important for nodule specific expression. One example is the soybean leghaemoglobin (lb) c3 promoter which were investigated in details in transgenic Lotus corniculatus plants. By analysing 5’ promoter deletions and hybrid promoters fused to the GUS and CAT reporter genes the following elements were identified in the lbc3 promoter; A strong positive element, a weak positive element, an organ specific element and a negative element (Figure 1, Stougaard et al. 1990).