ERBB-3 BINDING PROTEIN 1 acts downstream of TARGET OF RAPAMYCIN regulating multiple aspects of protein synthesis in the meristem and counteracts RETINOBLASTOMA RELATED to sustain cell growth. The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

The main function of the E2FB transcription factor is to restrict cell proliferation and establish quiescence during Arabidopsis leaf development; it acts in complex with RETINOBLASTOMA-RELATED. Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in e2fb mutant and overexpression lines during leaf development in Arabidopsis (Arabidopsis thaliana), we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels, either by expression driven by its own promoter or ectopically together with DIMERIZATION PARTNER A, further elevate the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in e2fb mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression, leading to reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number.

In the search for time- and tissue-specific promoters an RNA fingerprinting technique called cDNA-AFLP was used. A transcript derived fragment (TDF511) was isolated which showed high similarity to alcohol dehydrogenases. The gene corresponding to this TDF, named Stgan, is likely to be involved in biosynthesis or breakdown of compounds affecting gibberellic acid (GA) levels in the plant [Plant J. 25(6) (2001) 595]. In this article the isolation and characterization of a Stgan promoter region is reported. The promoter region of this gene was fused to a reporter gene encoding beta-glucuronidase (GUS) and introduced in potato plants. GUS staining was detected uniquely in stolon tips and nodes. RNA in situ hybridization experiments revealed that this gene was specifically expressed in parenchyma cells, in the stolon cortex. Comparison of this promoter sequence with several promoter databases resulted in the identification of several potential binding sites for transcription factors. From the in vitro-culture experiments Stgan transcription appears to be induced by long days, sucrose and different hormones such as gibberellic acid, ancymidol, ethylene and cytokinins.

Using cDNA-AFLP RNA fingerprinting throughout potato tuber development, we have isolated a transcript-derived fragment (TDF511) with strong homology to plant steroid dehydrogenases. During in vitro tuberization, the abundance profile of the TDF shows close correlation to the process of tuber formation. However, when tuberization is inhibited by the addition of gibberellins (GAs) to the growth medium, the appearance of TDF511 in the fingerprint is delayed, then steadily increases in intensity during later stages of development. TDF511 was used to isolate the corresponding cDNA (CB12). The DNA and deduced amino-acid sequences of the cDNA show high homology to a fruit-ripening gene from tomato, a series of steroid dehydrogenases, and the maize Ts2 gene. A section of the cDNA was cloned in antisense orientation behind a 35S CaMV promoter and transformed into potato. Transgenic plants expressing the antisense gene showed significantly earlier emergence, an increase in height, and longer tuber shape. In vitro tuberization experiments reveal extended stolon lengths in comparison to the controls. The analysis of endogenous GA levels showed that the transgenic antisense plants have elevated levels of biologically active GAs and their respective precursors. We propose that this gene plays a role in the metabolism of plant-growth substances important for tuber life cycle and plant development.

This paper shows that lipo‐oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection‐related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host‐specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

The Rhizobium-legume interaction results in the formation of a new plant organ, the root nodule, in which the bacteria are able to reduce atmospheric N2. These root nodules have a very distinctive tissue organization, which allows the N2 fixation process to occur [1]. In short the Rhizobium-legume interaction involves the following steps: It starts with the deformation and curling of root hairs. The rhizobia then invade the plant by means of a newly formed tube, called the infection thread. Meanwhile, cells in the root cortex start to divide and form the nodule primordium. Infection threads enter individual primordium cells, and bacteria are relased form the infection thread into the cytoplasm of the plant cells. The primordium cells then differentiate into the tissues that make up the mature nodule. Rhizobium thus induces three distinct developmental processes in its host (i) root hair deformation, (ii) infection thread formation, and (iii) induction of cell division followed by differentiation into the different nodule tissues.

During the last few years we have isolated early nodulin clones of both pea and soybean. In this chapter we will summarize the characteristics of these clones (Table 1). Furthermore we will describe how we started to use early nodulin clones to isolate Rhizobium signal molecules.

Here we demonstrate that Rhizobium mRNAs can efficiently be detected in developing root nodules with the in situ hybridization technique. We have been able to localize the Rhizobium nifH mRNA as well as the transcript of the regulatory nifA gene. Therefore, we expect that the in situ hybridization technique can generally be applied to locate Rhizobium mRNAs in root nodules. In pea nodules, the nifA and nifH mRNAs are first detectable in the third to fourth cell layer of the late symbiotic zone. In these cell layers, these mRNAs are detectable immediately at maximal levels. In older parts of the late symbiotic zone, the level of nifH mRNA remains constant, whereas the level of nifA mRNA decreases. Finally, the pattern of nif mRNA accumulation was compared with that of nodulin mRNAs.

The pea cDNA clone pPsENOD12 represents a gene involved in the infection process during Pisum sativum L.-Rhizobium leguminosarum bv. viciae symbiosis. The ENOD12 protein is composed of pentapeptides containing two hydroxyprolines. The expression of the ENOD12 gene is induced in cells through which the infection thread is migrating, but also in cells that do not yet contain an infection thread. Soluble compounds from Rhizobium are involved in eliciting ENOD12 gene expression. Rhizobium common and host-specific nodulation genes are essential for the production of these compounds. Two ENOD12 genes are expressed in nodules and in stem tissue of uninoculated plants. The gene represented by the cloned ENOD12 mRNA is also expressed in flowers, but a different transcription start may be used.

Recognition of the appropriate legume and nodule induction are controlled by common (nod) and host-specific nodulation (hsn) genes in Rhizobium. The nod and hsn genes are activated by the product of the regulatory nodD in conjunction with specific flavonoids excreted by the plant. Differences in the flavonoid specificity of the NodD proteins occur between different Rhizobium species, or between strains of a given species or even within one strain containing several copies of the nodD gene. Accordingly, the nodD gene controls the host-specific expression of nod and hsn genes. In addition, the nodulation genes are under not only positive but also negative regulation which is mediated by a nod-specific repressor protein. This dual control is required for optimal nodulation of the plant host. Further steps in nodule development are again controlled by the infecting Rhizobium. It was found that at least four different classes of Rhizobium fix genes are involved directly or indirectly in the expression of late nodulin genes, finally leading to the establishment of nitrogen-fixing symbiosis.Key words: Rhizobium meliloti, nodulation genes, plant signals, fix genes, alfalfa.

We have identified a nodD gene from the wide host‐range Rhizobium strain MPIK3030 (termed nodD1) which is essential for nodulation on Macroptilium atropurpureum (siratro). Experiments with nodA–lacZ gene fusions demonstrate that the MPIK3030 nodD1 regulates expression of the nodABC genes. Additionally, we used nodC–lacZ fusions of Rhizobium meliloti to show that the MPIK3030 nodD1 gene induces expression of these fusions by interacting with plant factors from siratro and from the non‐host Medicago sativa (alfalfa). The R. meliloti nodD genes, however, only interact with alfalfa exudate. In line with these results, no complementation of MPIK3030 nodD1 mutants could be obtained on siratro with the R. meliloti nodD genes, while the MPIK3030 nodD1 can complement nodD mutants of R. meliloti on alfalfa. Furthermore, R. meliloti transconjugants harbouring the MPIK3030 nodD1 efficiently nodulate the illegitimate host siratro. When compared with other nodD sequences, the amino acid sequence of the MPIK3030 nodD1 shows a conserved aminoterminus, whereas the carboxy‐terminus of the putative gene product diverges considerably. Studies on a chimeric MPIK3030/R. meliloti nodD gene indicates that the carboxy‐terminal region is responsible for the interaction with plant factor(s) and may have evolved in different rhizobia specifically to interact with plant–host factors.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, −B and −C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.

A 47-base-pair (bp) conserved sequence in the 5'-flanking regions of three transcriptional units coding for nodulation functions (nodABC, nodEFG, and nodH) has been identified in Rhizobium meliloti strain 41. The conserved region contains subsequences of 7 bp, 5 bp, and 25 bp. The conserved 25-bp sequence was synthesized and used as a hybridization probe; three additional copies of the sequence were identified in R. meliloti 41; all three were localized in the 135-kb nod/nif region of the symbiotic megaplasmid. Nucleotide sequence analysis of the six regions revealed that all contained the 47-bp conserved sequence but, with one exception, adjacent DNA regions did not have long conserved DNA stretches. The position of the 47-bp region was about 200-240 bp upstream of the translational start codons of the three nod genes. This conserved sequence is present in several other Rhizobium species and located adjacent to nod genes. We have demonstrated the involvement of this sequence in the expression of nodulation functions, which suggests that these extended promoter regions may have a role in the coordinated regulation of nodulation genes.