The symbiotic interaction between leguminous plants and rhizobia results in the development of a novel organ on the plant roots, the nodule, where bacteria provide fixed nitrogen for the host. Briefly, the microsymbionts attach to and enter the root hair cells while the plant forms a tube-like infection thread through which the bacteria move into the root cortex. Simultaneously, cortical cells are induced to divide and the infection threads invade these dividing cells forming the nodule primordium. This primordium differentiates into a mature nodule where rhizobia are converted into bacteroids and start to fix nitrogen (Cohn et al. 1998). Rhizobia induce nodule morphogenesis on the plant through the production of nodulation signals (Nod factors; Schultze, Kondorosi 1998). These Nod signals trigger the earliest stages of nodule development, including root hair deformation and curling, cortical cell division and the expression of several early nodulin (enod) genes. Under starvation for combined nitrogen, plants of certain Medicago cultivars have the capacity to form root nodules even in the absence of rhizobia (NAR+ phenotype), the so-called spontaneous nodules indicating that the developmental program of nodule formation preexists in legumes. These nodules show histological features similar to those of bacterium-induced nodules but the central zone cells contain only amyloplasts (Truchet et al. 1989). Hence, it has been suggested that spontaneous nodules might serve as carbon storage organs induced during nitrogen starvation and be the ancestors of the nitrogen-fixing nodules. Thus, Nod factors act as primary morphogenetic signals triggering a nodule developmental program although other plant factors are likely required for the regulation of nodule morphogenesis such as plant hormones (Fang, Hirsch 1998; Heidstra et al. 1997; Penmetsa, Cook 1997), the stele factor, uridine, (Smit et al. 1995) and the metabolic status of the plant (high carbon and low nitrogen) (Bauer et al. 1996).

The carbon metabolism of the plant host is adapted to altering demands during nodule development. Initially, amyloplast deposition is observed in the actively dividing cortical cells of the nodule primordia (Ardourel et al. 1994), demonstrating the accumulation of carbon translocated from the leaves. Later, the assimilation of symbiotically fixed nitrogen in the functional nodule requires a complex interplay with the carbon metabolism (Schultze, Kondorosi 1998) in order to satisfy (a) the demand of bacteroids for carbon and energy (required for nitrogen fixation), and (b) the provision of C4 carbon skeletons for the assimilation of fixed ammonia in the plant cells. The nodule primordium acts then as a sink and the plant tightly regulates nodule initiation through factors from the aerial parts of the plant ("shoot factor") (for review Caetano-Anollés, Gresshoff 1991), a phenomenon called autoregulation. The metabolic state of the root may be sensed by the plant, whereby intermediates may act directly as signals to modulate cellular responses or indirectly by influencing the activity of internal plant hormones required for nodule initiation or starch deposition.

Plasmodesmal (PD) function seems to be responsible for cell-to-cell communication and signaling in plant development and there is a growing body of evidence that a complex supracellular communication network acting through PD is present in plants (Lucas, Wolf 1999). This network was originally identified by studying viral movement between cells. Viral movement proteins (MPs)

are localized to PDs and affect their function as first explored by expression of the MP gene in transgenic plants (Deom et al. 1990; Wolf et al. 1991). Based on the assumption that altering plasmodesmal size exclusion limit should affect the transport rate of small molecules, including sucrose (Tyree 1970), during the last few years, carbon transport and allocation in transgenic tobacco plants expressing the TMV-MP have been studied (Lucas et al. 1993; Olesinski et al. 1996). These data allowed the advancement of a hypothesis that trafficking of regulatory (information) molecules, through plasmodesmata may establish a special supracellular communication network regulating carbon partitioning. Plants may exploit this to create specialized physiological and developmental domains where signaling may occur during the formation of an organ primordium (e.g. the nodule initials) or between different cell layers of a meristem (Lucas, Wolf 1999 and references therein). Molecular mechanisms involved in the control of nodule organogenesis in the plant host are poorly understood. In our laboratory, several approaches are being attempted to understand how cell-to-cell communication processes may interact with key regulatory genes to understand the complex process of nodule development. In this paper, we describe the preparation of M. truncatula transgenic plants to characterize cell-to-cell communication processes and various functional approaches carried out on selected regulatory genes to understand their role in nodulation.

2. Procedures

Preparation of transgenic Medicago truncatula plants and RT-PCR experiments were done as described (Charon et al. 1999; Frugier et al. 2000). Western analysis was carried out according to standard techniques (Wolf et al. 1991). Bombardment of germinating alfalfa roots using "microtargeting" of DNA constructs has been described (Sousa et al. 2001).

3. Transgenic Plants Expressing Viral Movement Proteins and GFP Fusion Proteins Can Be Used to Study Cell-to-Cell Communication in Legumes

We have prepared diverse transgenic M. truncatula plants which may serve to analyze carbon partitioning during the symbiotic interaction. First, various independent transgenic lines expressing the movement protein of TMV (tobacco mosaic virus) were characterized. This MP has been shown to affect carbon partitioning in transgenic potato plants (Lucas, Wolf 1999). TMV-MP expression driven by the 35S promoter was confirmed using Western analysis of leaf extracts. No obvious visible phenotype was observed on these plants when growing under normal conditions or in symbiosis with Sinorhizobium meliloti. We could not find any significant differences in the number of nodules and further work is being carried out to analyze in detail the early steps of the interaction, such as primordium formation and amyloplast accumulation in dividing cortical cells. Second, we have expressed the CMV-MP (from cucumber mosaic virus) fused to GFP in M. truncatula. Selected plants with high levels of CMV-MP-GFP expression showed a dotted pattern of fluorescence corresponding to the labeling of PDs in various cell types. Third, plants containing phloem-specific expression of GFP have been characterized (using a Atsuc2-GFP construct, kindly provided by Dr N. Sauer). In these plants, phloem unloading of GFP into carbon "sinks" such as lateral roots and organ primordia was followed (see Imlau et al. 1999), by monitoring green fluorescence in tissues. These two latter transgenic lines are excellent tools to monitor in vivo the process of phloem unloading, carbon sink formation and nodule initiation.

4. The Role of enod40 in Nodule Organogenesis

In addition to this physiological work, we have characterized several regulatory genes induced during nodule organogenesis in Medicago species using various approaches (Crespi et al. 1994; Frugier et al. 1998). One of the earliest nodulin genes associated to the nodule developmental program is enod40. In response to bacterial inoculation of the roots, enod40 transcripts are detected first in the root pericycle opposite to the protoxylem pole, then in the dividing cortical cells and in all differentiating cells of the growing nodule primordia (Fang, Hirsch 1998 and refs therein). We have shown that, under nitrogen-limiting conditions, overexpression of this gene resulted in a significant increase of cortical cell divisions in M. truncatula roots (Charon et al. 1997). This was accompanied by a high level of accumulation of amyloplasts in the dividing cells. During nodulation, overexpression of enod40 modified the early stages of development and induced proliferation of cortical cells all around the cortex in the infected region close to the root tip. This suggested that (a) enod40 induction in the cortex may be a determinant of nodule initiation (Charon et al. 1999), and (b) its primary function is not exerted directly on triggering cell division per se. In contrast to auxin, enod40 does not have the capacity to induce cell proliferation irrespective of the nitrogen status of the plants or the root position. In addition, a putative co-suppression phenomenon induced on selected lines yielded plants showing arrested nodule development, indicating that enod40 expression is required for appropriate development of the primordium (Charon et al. 1999). Due to its expression pattern in vascular tissues and nodule initials, as well as to the phenotype of the transgenic plants, a role of enod40 in cell-to-cell communication between the vascular tissue and specific cells of the cortex to allow proper organization of the nodule primordium seems very likely.

The enod40 genes (Cohn et al. 1998; Schultze, Kondorosi 1998) are very peculiar because they code for about 0.7 kb RNAs containing only short ORFs. Modeling predicts that enod40 RNA sequences have the tendency to form particularly stable secondary structures, a property shared with several biologically active RNAs (Crespi et al. 1994). On the other hand, a very small ORF corresponding to 10 to 13 amino acids in the 5' end of the transcripts is common among these genes and has been proposed to be the active gene product (van de Sande et al. 1996). Recently, we have demonstrated that several small ORF (sORFs) of enod40 were translated when fused to a reporter gene. In addition, microtargeting of Mtenod40 into Medicago roots induced a cell-specific growth response, division of cortical cells, that was used to test different gene derivatives containing specific point mutations and deletions (Sousa et al. 2001). These experiments indicated that translation of two sORFs present in the conserved 5' and 3' enod40 regions was required for activity. Moreover, deletion of a Mtenod40 region present in between the two sORFs and spanning the predicted RNA structure showed low activity in our assay, without affecting translation of the sORFs. Even though the encoded sORF-peptides maybe the functional gene products, the structured RNA region also participates in gene regulation. These data revealed that a complex cellular mechanism may be implicated in the translation and primary cellular function of enod40.

5. A Vascular Krüppel Transcription Factor Involved in the Formation of the Symbiotic Zone

Another regulatory gene identified was a Kriippel-like Zn-finger gene, Mtzpt2-1. This gene is strongly expressed in vascular bundles of roots and nodules, and antisense plants grew normally but developed Fix- nodules where differentiation of the nitrogen-fixing zone and bacterial invasion were arrested. These results indicate that a vascular bundle-associated Krüppel-like gene is required for the formation of the central nitrogen-fixing zone (Frugier et al. 2000). Interestingly, a homologous gene was shown to confer salt tolerance to yeast cells. Indeed, Mtzpt2-1 was also able to induce this response in yeast. Moreover, this transcription factor is strongly and rapidly induced after application of salt stress to root and nodules, suggesting that it may participate in osmotic stress responses in plant cells. Therefore, we think that Mtzpt2-1 may be involved in the osmotic adaptation of the nodule vascular tissues to support nitrogen fixation.

6. Novel Kinase Associated to the Initial Steps of the Symbiotic Interaction

Very few regulatory kinases associated to nodulation are known. A gene, Mtpkl, encoding a novel protein kinase containing an ankyrin domain was identified as being induced during nodulation and in spontaneous nodules (Frugier et al. 1998). We have made translational fusions of the entire Mspkl gene to GFP in order to localize the protein in root tissues during symbiosis and in transfected onion cells. This kinase seems to co-localize with microtubules in the latter cells and may be associated to the microtubule rearrangements required during Rhizobium infection.

Expression of this gene was detected in different alfalfa organs and in the early stages of the symbiotic interaction.

The Mtpkl gene was isolated by screening of an M. truncatula BAC library and the sequence of its genomic region and of certain adjacent clones were determined. Several genes showing homologies to previously identified sequences in databanks were identified in the vicinity of the Mtpkl gene. The distribution of exons and introns was analyzed in detail for Mtpkl and compared with three homologous genes identified in Arabidopsis thaliana. These data suggest that Mtpkl may be involved in the early steps of the symbiotic interaction, though it is not exclusively associated with nodulation. The developed tools will serve to analyze the possible function and localization of Mtpkl during the initiation of nodule organogenesis.

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