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Surprising developments in the legume root nodule Douglas Cook

Summary

Root nodule morphogenesis involves the induction of mitotic activity in otherwise quiescent root cortical cells, giving rise to the nodule primordium. One gene expressed during nodule initiation, ENOD40, has been implicated in nodule growth and/or differentiation(’ v 2 ) . Interestingly, although the nucleotide sequence of €NOD40 genes from ~ o y b e a n ( ~ , ~ ) and Medicago(’ 12) are highly homologous, they are unlikely to encode a similar protein product. In fact, a remarkable feature of these genes is their apparent lack of protein coding potential. Thus, €NOD40 is a member of the growing list of eukaryotic genes whose RNA product is implicated in control of cell growth and differentiation, the so called riboreg~lators(~).

Introduction: nodule morphogenesis The legume root nodule is a specialized plant organ where nodule-inducing bacteria, rhizobia, and their legume host conduct symbiotic nitrogen fixation. Nodule morphogene- sis is initiated by a molecular dialogue between the plant root and compatible soil rhizobia. Briefly, flavonoids or cer- tain other metabolites excreted from the plant root induce the expression of bacterial nod genes@), whose function is required for nodulation and synthesis of a bacterial signal molecule, a lipo-oligosaccharide called Nod factor(7). Puri- fied Nod factor can induce formation of nodule primordia in a host-plant-specific manner, such that variable chemical substituents on a common Nod factor backbone deter- mine the biological specificity of the Nod factor signal mol- ecule and the host range of the respective bacterium(7).

Morphogenesis of the nodule organ commences when compatible rhizobia encounter developing root hair cells, and Nod factors produced by the prospective symbiont trigger two nearly coincident responses in the plant root: (1) distortion of root hair cell growth(8), and (2) mitotic acti- vation of the subtending cortical cells producing the nodule primordium(9). While bacteria invade the root tissues, the distal cells of the primordium organize to form a meristem and ultimately the various nodule tissues. Because root cortical cells are induced to divide prior to bacterial infec- tion, nodule morphogenesis and bacterial infection are likely to involve separate processes. In fact, several lines of evidence indicate that the genetic blueprints for nodule morphogenesis are contained entirely within the plant and

that the bacterium’s role in organogenesis is to trigger the plant’s developmental program. The most compelling evi- dence for this view is that selected genotypes of alfalfa can develop nodule structures spontaneously, without a requirement for the bacterium or the bacterial signal(’O).

€NOD40 expression is associated with mitotically active cells ENOD40 is one of the few plant genes known to be expressed at the onset of nodule morphogenesis. Prior to the first observed cell division events, €NOD40 transcript is induced in the root pericycle opposite incipient nodule primordia, and then in the nodule primordium This pattern is largely coincident with the spatial induction of cell cycle genes during nodule initiation in alfalfa(g), indi- cating that mitotically activated cells are a primary locus of early €NOD40 induction. The correlation between €NOD40 induction and cell division is strengthened by the observation that non-symbiotic expression of €NOD40 is also associated with mitotically competent plant cells, including pre-emergent lateral root tips, the margins of young leaf primordia, the stem procambium, and actively dividing tissue culture cells(l r2).

Two types of nodule ontogeny can be distinguished based on the duration of mitotic activity in the meristem: Medicago spp. possess a persistent nodule meristem, so- called indeterminate nodules, while soybean nodules pos- sess transient meristem activity, so-called determinate nodules. In most respects €NOD40 expression is similar

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in these two nodule types, particularly the association with mitotically active cells during nodule morphogenesis and with pericycle cells of the vascular bundle in mature nod- ules. A primary difference, however, is the continued expression of €NOD40 in the meristem region of mature Medicago nodules(lT2); a similar expression pattern is lack- ing in soybean nod~ les(~3~) due to the absence of a compa- rable tissue. Nevertheless, ENOD40 transcript continues to be present at a high level in mature soybean nodules; part of this can be accounted for by expression in the vas- cular pericycle, but ENOD40 also appears to be expressed in the uninfected cells of the nodule central tis-

The association of €NOD40 transcript with this ter- minally differentiated tissue indicates that, at least in the determinate soybean nodule, mitotic activity is not a uni- form feature of cells that express ENOD40. A role in nitro- gen transport has been suggested for the uninfected cells of the soybean nodule central tissue(”), prompting the suggestion that ENOD40 might be involved in processes associated with nutrient transport(3).

Several lines of evidence indicate that ENOD40 expression is controlled by the plant’s program for nodule morphogenesis. First, bacterial infection is not a prerequi- site for €NOD40 induction because exopolysaccharide mutants of the bacterium, which induce nodule morpho- genesis without persistent bacterial infection, also induce €NOD40 expression(2). Nodules induced by exopolysac- charide mutants possess a poorly defined, minimally active meristem(12), and correspondingly contain reduced levels of €NOD40 transcript. Second, induction of nodule primordia by the exogenous application of mitogenic agents, such as Nod factor or the auxin transport inhibitor NPA (1 -napthy1 phthalamic acid)(I3), results in the coinci- dent induction of €NOD40 expression. For example, Nod factor induction of ENOD40 is effective only at concentra- tions that also induce cortical cell division(’); lower concen- trations of Nod factor (4 0-9 M ) , which effectively induce other host responses including root hair deformation and expression of certain nodule-specific genes(14-16), fail to stimulate either cortical cell division or €NOD40 expression. Thus, €NOD40 induction by Nod factor is cor- related with reactivation of the cell cycle in the root cortex. The ability of the auxin transport inhibitor, NPA, to induce nodule-like primordia(13) and €NOD40 expressiod2) suggests that both events may be mediated by changes in the concentrations of endogenous growth regulators (i.e. auxin and cytokinin hormones). This view is supported by the observation that €NOD40 and nodule-like structures are induced by treatment with cytokinin hormone~(~9’~). Finally, €NOD40 is also induced in nodules that arise spontaneously on selected alfalfa genotypes(’), without a requirement for the bacterium or Nod factor. Thus,

ENOD40 expression appears to be part of a plant-con- tained program for nodule organogenesis.

ENOD40 may function in nodule growth and development, but is unlikely to code for a protein product In the absence of phenotypic data, the functions of many early nodulation genes have been inferred largely on the basis of their patterns of expression during nodule devel- opment. For ENOD40, the physical correlation of tran- script with sites of active cell division suggests that the €NOD40 gene may be either involved in or directly influ- enced by events related to plant growth and differentiation. Further circumstantial evidence for this conclusion was provided by overexpression and antisense studies with an €NOD40 transgene”). Agrobacteriurn transformation of alfalfa explants with an overexpressing €NOD40 trans- gene produced teratomas, characterized by polyembryo- genic calli that failed to regenerate fully developed plants. In contrast to both control and overexpressing constructs, explants infected with an ENOD40 antisense construct exhibited impaired callus growth and did not yield embryos. These results implicate €NOD40 as an impor- tant determinant of plant growth and/or development.

Despite indications that ENOD40 may have a functional role in nodule growth and/or differentiation, €NOD40 tran- scripts appear unlikely to encode a protein product. Align- ment of the Medicago(’I2) sequences with those of soy- beat^(^,^) indicates that although they share considerable nucleotide sequence identity (near 80%), and they are all polyadenylated, they do not possess a significant common open reading frame. In fact, each of the €NOD40 tran- scripts are characterized by the presence of many short open reading frames, none of which possess strong homology to known genes or proteins. Furthermore, esti- mates of coding probability based on third position nucleotide usage were low for all of the €NOD40 homo- logues. In M. truncatula the longest open reading frame of 28 amino acids is located downstream of five AUG codons and fifteen translation termination signals, features that are likely to impair translation(18). Alternative translation start sites (CUG, GUG or ACG) were not found in the Med- icago genes, and in vitro translation from Medicago tran- scripts could not be detected(’). In soybean, a low level of in vitro translation was detected by western blot analysis using a soybean ENOD4lXaMV PI translational fusion and antibodies directed against PI epitoped3). However, the predicted translation product originated at a non-con- ventional start codon, and the implicated soybean open reading frame is missing from the Medicago sequences. €NOD40 transcripts have been localized to the cyto- plasm(lr2), specifically in the monosome fractiod2).

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Although this subcellular localization is indicative of non- translated mRNAs, it remains possible that €NOD40 is translated to yield a short peptide, and certain plant pep- tides are known to affect large changes in plant gene expression(1g). However, it is difficult to reconcile the lack of a common open reading frame between the soybean and the Medicago genes, in particular because they are presumed to mediate the same processes. Furthermore, both the Medicago and soybean ENOD40 transcripts pos- sess relatively low estimated free energies of folding(l), a feature typical of other biologically active non-coding RNA molecules, but not common among translated RNAs.

Perspectives Recently, several other eukaryotic RNAs have been iden- tified that are implicated in processes related to growth and development, yet which have a low probability of cod- ing for a protein product. These genes include H7aZ0), Xisd2I) and H i ~ - 7 ( ~ ~ ) , all of which share several features in common with fNOD40. First, they are all polyadenylated and contain a high density of terminators in all open read- ing frames. Second, homologues of these genes, which have been sequenced from different animal species, are highly conserved at the nucleotide sequence level, yet exhibit little conservation between their multiple small open reading frames, making it unlikely that they encode functional proteins. In fact, no protein product has been detected for any of these genes, and in cell fractionation studies their transcripts were not associated with the polysomal fraction. Third, all of these genes are develop- mentally regulated. The H79 gene has been implicated in mammalian embryo development, while the Xist gene has been implicated in X-chromosome inactivation, and the murine His-7 gene has been implicated in the neoplastic process. In the case of the H79 gene, ectopic expression caused prenatal lethality in mice(23), and suppression of growth and development in murine embryonal tumor cell I in es(24).

A common characteristic of non-coding regions of RNA is a high frequency of stop codons and a lack of extensive open reading frames. Partly on this basis, such non-cod- ing regions of RNA have been assumed to lack significant biological function. However, this paradigm is slowly erod- ing as RNA molecules are implicated in such fundamental biological processes as protein synthesis(25) and RNA splicing(26). In addition, the 3’ untranslated region of cer- tain transcripts has been shown to act in cis to affect mRNA function(27), and in trans to regulate transcription of developmentally related genes(28). The recent observation that entirely non-coding transcripts, such as €NOD40 and H79, may function directly to affect growth and differen-

tiation adds to the growing paradigm that RNA molecules can directly catalyze biological reactions, and may have important implications for development in both plants and animals.

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Douglas R. Cook is at the Texas A&M University, College of Agriculture and Life Sciences, Dept of Plant Pathology and Microbiology, College Station, TX 77843, USA.

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