Biochemical and molecular studies of symbiotic nitrogen fixation

Biochemical and molecular studies of symbiotic nitrogen fixation Frans I. de Bruijn and I. Allan Downie MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, USA and John Innes Institute, AFRC Institute of Plant Science Research, Norwich, UK. Significant advances in the area of symbiotic nitrogen fixation include the identification of a rhizobial factor responsible for nodule induction and new insights into nodulin gene regulation, as well as the elucidation of a signal transduction pathway involved in nitrogen fixation gene control. There have also been reports about the nodulation of non-legumes such as rice, wheat and oilseed rape. Current Opinion in Biotechnology 1991, 2:184-192

Introduction Biological nitrogen fixation constitutes one of the most essential global processes. Approximately 170 million tons of atmospheric nitrogen are converted into ammonia every year and of this, 120 million tons are contributed by free-living, associative and strictly symbiotic nitrogen-fixing microbes. The symbiotic interaction between members of the Rbizobiaceae and leguminous plants accounts for 80% of the stably fixed nitrogen [1], as it results in the direct incorporation of nitrogen into plant amino acids and proteins. This is in direct contrast to the ammonium produced by free-living diazotrophic bacteria, which can be rapidly lost by various denitrification reactions in the biosphere. Symbiotic nitrogen fixation is carried out in specialized organs (nodules) whose formation is induced on their plant host by rhizobia. A variety of bacterial and plant genes are specifically activated during the process of nodule ontogeny and also in mature, nitrogen-fixing nodules. Multiple signals involved in nodule-specific gene expression go back and forth between the symbiotic partners. Moreover, the infecting rhizobia need to differentiate into a distinct form, the bacteroid, in order to fix nitrogen for the plant's benefit [2,3-]. During the last 20 years, considerable interest has developed in improving the efficiency of symbiotic nitrogen fixation, as well as in more speculative ideas, for example, the extension of the host range of rhizobia to agriculturally important crops, such as rice and wheat, or the transfer of the ability to fix nitrogen to plants themselves [4,5-]. The last approach would be extremely complex because of several intrinsic properties of biological nitrogen fixation [5"J. First, the reduction of dinitrogen (N 2) by the nitrogenase enzyme complex is an extremely energy-intensive process, requiring up to 42 moles of ATP per mole of N2 reduced [6]. Second, the nitrogenase enzyme is extremely oxygen-sensitive. Third, multi-

pie genes (up to 21 in the diazotroph Klebsiella pneumoniae [7]) are involved in the process of nitrogen fixation. Therefore, recent efforts have concentrated on the first two approaches. A number of fundamental questions will have to be answered before the possibility of either improving the efficiency of symbiotic nitrogen fixation or extending its host-range can be considered. Here, we will highlight some of the recent progress made in addressing these questions.

Rhizobial signals involved in nodule induction The recognition between leguminous plants and the specific rhizobial strains that nodulate them is mediated primarily by low-molecular-weight signal molecules. The initial steps in the plant-microbe interaction essentially involve two stages of signalling. In the first stage, the legume roots secrete specific flavonoids or isoflavonoids which are recognized by the rhizobia [2]. Subsequently, a group of rhizobial nodulation genes (nod, nol) are induced (Fig. 1) and their products are involved in sending host-specific signals, which are recognized by the appropriate legume hosts. Of particular interest is a secreted glycolipid molecule, synthesized by some of the nod gene products. This molecule induces root hair curling (which constitutes the first observed plant response to rhizobial infection), as well as cortical cell division, and it also triggers a program leading up to nodule ontogeny [2,3"]. The structure of one of these signal molecules, synthesized by Rtaizt~ bium meliloti (symbiont of alfalfa or Medicago species), has recently been elucidated (Fig. 1) [8"]. This molecule is unique amongst the signal molecules involved in plant cell division or differentiation that have been identified to date, because it induces a plant response at an astonishingly low concentration (10 -11M), is highly specific for those legumes nodulated by ~ meliloti, and is capable of

Abbreviations Ib~leghernoglobin; GUS--~-glucuronidase. 184

(~ Current Biology Ltd ISSN 0958-1669

Biochemical and molecular studies of symbiotic nitrogen fixation de Bruijn and Downie

(b)

...g~ 1 2----0 OH~'

NH

co= ]

1

,&-~Y' 'L--~o

,.L'i" '2~'o

&-.~i ,2__ o

JC°CH3)l

OH~I

OFI~I

OH'l

~L

Nit

COCH3

NIH

COCH 3

CH

,// HC I

(CIH2)s CH

II CH I

(CH2)5 I

CH3

initiating the complete developmental program required for nodule formation [8"',9"]. The groundwork leading to the identification of this compound was laid several years ago [10], when it was found that a low-molecular-weight compound with root hair curling activity was present in the rhizosphere of Rbizobium-infected legumes. Using the same root hair curling response as a bioassay, the signal molecule NodRm-1 [8-,] was purified from filtrates of cultures of t~ rneliloti which had been induced to express the nod genes. Although the NodRm-1 factor shown in Figure 1 is highly specific for legumes which are normal hosts for R rneliloti, it is clear that distinct, but closely related, signal molecules must be made by other rhizobial strains. It has been established that the nodulation genes nodABC (Fig. 1) are required for root hair curling and the synthesis of the NodRm-1 factor [8"',11"]. These genes are highly conserved in all rhizobial species [2] and therefore may be involved in the formation of a 'core structure' (e.g. the acylated polyglucosamine shown in Fig. 1), which is then chemically modified by other, host-specific nod/nol gene products to become a host-specific signal. This hypothesis is supported by the finding [11.] that mutations in the host-specific nodH or nodf2 genes of

NH

I COCH 3

II I

Fig. 1. Organization of the ghizobium leguminosarum bv. viciae and R. meliloti nodulation (nod/nol/syr) genes and structure of the NodRm-1 symbiotic signal molecule. (a) The organization of the nodulation genes and their direction of transcription are indicated by black filled arrows. The identification and function of some of these genes are described in the text [2,8..,23.]. The R. meliloti nol FGHI genes were identified by N Baev, A Kondorosi and colleagues (personal communication) and the syrM gene (a nodD-like regulatory gene) by S Long and colleagues (personal communication). (b) The structure of the R. meliloti NodRm-1 signal molecule is a sulphated tetrasaccharide of D-glucosamine, in which three amino groups are acetylated and one substituted with a C16 bis-unsaturated acyl group [8"']. The acetyl group (bracketed) was identified during the analysis of the related R. leguminosarum bv. viciae signal molecule [21.°]. It appears that some signal molecules contain five sugar moieties rather than four [21oo]. The proposed roles of the nod gene products in the synthesis of the signal molecule are indicated by arrows (for details see text).

R meliloti resuk in the formation of a signal molecule that can be recognized byvetch (a normal host for R leguminosarum by. viciae), but no longer by alfalfa (a normal host for R meliloti). Moreover, when the nodHQ genes, which are normally absent from R leguminosarum bv. viciae (Fig. 1), are transferred into such a strain, the resulting transconjugant acquires the ability to produce the signal molecule allowing the nodulation of alfalfa [11. ]. It is now apparent that the nodHPQ genes are involved in attaching the sulphate moiety to the NodRm-1 signal molecule, as nodH or nodQ mutants produce a factor lacking this moiety (Fig. 1) [9"]. In fact, the nodPQ gene products have been found to be functionally equivalent to the Escberichia coli cysCD gene products that are involved in the formation of adenosine 5'-phosphosulfate, which is used in sulphate transfer reactions [12..]. A number of amino acid homologies have been detected between nodgene products and enzymes of known function. The nodF product has been found to share homology with acyl carrier proteins involved in fatty acid synthesis [13] and the nodE product has been found to resemble [3-ketoacyl synthases (condensing enzymes) [14]. These observations are of particular interest, because the nodFE genes have been shown to be key dete rminants of host-specificity [ 15,16" ]. Although different

185

186

Plantbiotechnology Rhizobium spp. have homologous nodFE genes, there is strong evidence to suggest that the genes are subtly different in their function [15,16-]. Another host-specificity gene (nodG) also encodes a protein with a homologne in fatty acid biosynthesis, namely the 13-ketoacyl-acyl-carrier protein reductase enzyme of avocado [17]. With regard to the NodRm-1 factor, it is likely that these gene products direct host-specificity by determining the nature of the acyl group attached to the glucosamine polymer (Fig. 1).

To date, no nod gene product has been identified which shows homology to acyl transferases and it is possible, therefore, that the transfer of the acyl group to the polyglucosamine is mediated by the product of a normal housekeeping gene. Altematively, one could speculate that the nodC gene product, which plays an enzymatic role in the synthesis of the factor (J Schmidt, M John and J Schell, personal communication), in addition to being a trans-membrane receptor-like protein [18], may be responsible for the acylation of the polyglucosamine moiety, synthesized through the concerted action of the nodAB gene products [8",19]. The product of the nodL gene had been found to share considerable homology with acetyl transferases, such as the E. coli lacA gene product [20]. It now appears that the nodL product is capable of attaching an acetyl group to the reducing end (Fig. 1) of an acylated polyglucosamine molecule produced by R leguminosarum bv. viciae [21..]. It is possible that this type of modification could also introduce specificity into this class of signal molecule. Secretion of significant amounts of N-acetyl glucosamine derivatives could pose a problem with regard to the intracellular pools of this important precursor of cell wall biosynthesis. This may be obviated by elevated levels of the nodal gene product, which shares considerable homology with E. coli glucosamine synthase and can direct the formation of glucosamine (C Marie and JA Downie, unpublished data; N Baev and A Kondorosi, personal communication; Fig. 1). Thus, rhizobia have a set of genes involved in the synthesis and host-specific modification of a (core) signal molecule, which is essential for root-hair curling and the early stages of nodule ontogeny. In addition, other types of signal molecules may be essential (see below) and other nod/nol genes may play an important role in symbiotic interactions between Rhizobium and plants [2]. For example, the R leguminosarum bv. viciae nodO gene encodes a calcium-binding protein, that is secreted [22,23.] and plays an important but as yet undefined role In infection [24.]. It is clear that one of the most crucial future tasks will be the identification of the receptors for the various rhizobial signal molecules. Receptors, such as the one interacting with the NodRm-1 factor, must be exceedingly sensitive, considering the concentration at which these signals evoke the plant response [8..,11. ] and must be able to distinguish between closely related signal molecules. Plant root lectins have been suggested to be part of the Nod signal/plant receptor complex [8.,]. This notion is backed up by the intrigu-

ing observation that transgenic clover roots, harbouring the pea lectin gene [25], acquire the ability to be nodulated b y / ~ leguminosarum bv. viciae (which normally nodulates pea but not clover). In any case, the characterization of the NodRm-1 factor is an essential step towards the identification of a plant receptor, which may be unique in several respects, and towards elucidating the signal transduction pathway involved in this highly evolved prokaryote-eukaryote interaction.

Expression of plant genes involved in nodule formation and symbiotic nitrogen fixation Plant genes which are specifically induced during the formation of a nitrogen-fixing nodule have been called nodulin genes [26]. 'Early' nodulins are involved in the infection process, the formation of the nodule primordium and the differentiation of the nodule meristem into a nodule [3"]. 'Late' nodulins are expressed in the mature nodule, concomitant with the induction of the bacterial (bacteroid) nitrogen fixation process, and are involved in nitrogen fixation and assimilation, oxygen transport, carbon metabolism and specialized processes in the peribacteroid membrane [3",27]. Different early and late nodulin genes have served as outstanding molecular markers for distinct stages of the infection and nodule formation processes, as they are not only symbiotically induced, but also developmentally controlled and expressed in a tissue-specific fashion [3"]. For example, in pea nodules the early nodulin genes Enod5 and E n o d l 2 are expressed first during the infection process [28.,29..], whereas the other early nodulin genes Enod3 and Enodl4 are expressed maximally in the symbiotic zone [28.]. Expression of late nodulin genes, such as the leghemoglobin ( l b ) genes, can be detected in the infected cells of the late symbiotic zone [28.]. One additional early nodulin gene has been investigated in detail, the Enod2 gene. This gene has been found to be expressed in a completely different part of the developing nodule, namely in the nodule parenchyma (or inner cortex), where it has been suggested to be involved in creating an oxygen barrier for the infected zone [30"]. Thus, considerable knowledge has been gained about the developmental induction and tissue-specific expression of several plant genes involved in nodule formation. 4

Induction of nodulin genes Clearly, different signals are involved in early and late nodulin gene induction [2,3",31"]. E n o d l 2 gene expression is dependent on the rhizobial nod genes [29"], but can also be activated by application of the purified NodRm-1 factor to the roots [9"]. The Enod2 gene is expressed in the so called 'empty nodules' (lacking infection threads and bacteria) that are induced by mutant rhizobial strains lacking certain exopolysaccharides or [3-glucans [32,33"]. On the other hand, expression of the l b and other late nodulin genes has been found to require the physical presence of infecting bacteria or bac-

Biochemical and molecular studies of symbiotic nitrogen fixation de Bruijn and Downie

teroids inside infection threads or within the plant cell cytoplasm [3",27,31",32]. Although the exact signal transduction pathway is unknown, phytohormones have been shown to be involved in early nodulin induction. Enod2 expression has been found in 'pseudonodules', which have been induced on alfalfa roots by auxin-transport inhibitors [33",34] and in spontaneously formed nodules on the same plant host [35"]. Moreover, a cloned cytokinin (zeatin) biosynthesis gene has been found to be able to complement partially a nodABC-- mutant of t~ meliloti, resulting in empty alfalfa nodules in which Enod2 is expressed (S Long, personal communication) [36]. Lastly, the Enod2 gene of the tropical, stem-nodulated legume Sesbania rostrata [37] has been shown to be induced specifically and rapidly (within 2 h) by cytokinins at physiologically significant concentrations of 5-10 nM (C Dehio and F J de Bruijn, unpublished data) [31"]. This would also make the S. rostrata Enod2 gene the first example of a plant gene specifically regulated by cytokinin in the absence of other environmental control signals. Thus, the rhizobia appear to be manipulating endogenous signal transduction pathways in the plant in order to regulate cell division and differentiation, during the induction of a nodule [3"]. In the case of late nodulin genes, such as the lb and N23 genes, as well as the gene encoding the nodule-enhanced glutamine synthetase enzyme subunit (gln~,), highly conserved cB-acting elements have been delimited in their 5'-upstream regions. Studying the expression of chimeric reporter genes in transgenic plants [31",38",39",40,41"] has shown that these regions are responsible for the cellspecific expression of the genes in nodules or infected cells. In addition, nodule-specific and other tissue-specific transacting factors have been identified, that specifically interact with conserved AT-rich sequences and other binding sites in the promoter regions of these genes [31.,41.,42,43,44.]. It is expected that the different experimental approaches summarized above, starting from rhizobial signals in the direction of the plant response and working backwards from the promoter regions of the nodulin genes, will merge in the near future, to yield a better insight into the regulatory circuitry involved in nodule-specific plant gene expression.

Regulation of the rhizobial nitrogen fixation genes Oxygen supports the ATP production required for nitrogenase activity in the actively respiring bacteroids. However, it is also capable of severely inhibiting nitrogenase and repressing the synthesis of the nitrogen fixation (Nil/Fix) polypeptides [45]. Thus, the intracellular 02 concentration represents a very important signal for symbiotic nitrogen fixing bacteria, which have evolved a variety of circuitries to regulate their niffix genes accordingly [45]. The rhizobial niffix genes are induced only under microaerobic conditions. This represents the third mechanism known to deal with the 02 problem in the nodule, the others being the synthesis of highly efficient oxyhemoproteins (Lbs) that transport 02 to the

bacteroids at very low intracellular 0 2 concentration [46], and the 0 2 barrier in the nodule parenchyma [30"]. The regulation of the rhizobial n ~ f i x genes has been shown to be highly complex, invoMng a cascade control system consisting of multiple regulatory loci (Fig. 2) [47,48"]. In/~ meliloti, the niJTfix genes are controlled by two transcriptional regulators, encoded by the nt~7~land fixK genes, which in tum are regulated by the products of the fixLJgenes (Fig. 2) [49,50,51,52"]. The fixLJgenes belong to a family of two-component regulatory systems, consisting of a sensor component (FixL) and a transcriptional activator protein (FixJ) [47,50]. FixL has been suggested to be a trans-membrane protein, which senses the 02 concentration [50,52"] and then passes the signal on to FixJ. As had been observed for other two-component regulatory systems [47], the signal transduction pathway appears to involve phosphorylation of the regulator protein. In fact, FixL has been found to have kinase activity and to phosphorytate itself and subsequently transfer the phosphate group to FixJ (M Gillis-Gonzalez, G Ditta and D Helinski, personal communication) [53 °,]. Regarding the mechanism of O2-sensing by FixL, a very interesting observation has been presented recently, namely that FixL is an O2-binding hemoprotein and that the liganded state of the FixL heme moiety affects the kinase activity of the protein [53"]. The regulatory circuitry responsible for 02 control in/~ meliloti (Fig. 2) is not universal for all rhizobia [48.]. Moreover, another level of 0 2 control, acting directly on the NifA protein, has been reported for/~ meliloti [49,54] and Bradyrhizobium japonicum [55,56"]. It has been proposed that NifA is a metal-dependent gene regulator and that the metal-dependence may be related to its regulatory role in response to 02 [56"]. Rhizobial NifA proteins contain a putative metal-binding domain, with four essential cysteine residues [56*], although the kind of metal and its precise interaction with the protein remain to be elucidated. Thus, both a hemoprotein and a metal-binding protein appear to be involved in O2-regulation of the nijYfix genes in rhizobia and it could be speculated that oxidized or reduced iron molecules are responsible for the 02 response in both cases [53°',56"].

Nodulation of rice, wheat and oilseed rape Some non-leguminous plants are known to form nitrogen fixing nodules in response to infection with rhizobia (e.g. Parasponia) [57], and several non-legumes form nitrogen fixing symbioses with microbes of the genus Frankia [58]. Therefore, there is no a priori reason to assume that other non-leguminous plants do not have the genetic capacity to sustain a nitrogen fixing symbiotic interaction. However, as many non-legumes are not normally found to bear nitrogen-fixing nodules, it could be concluded that a certain barrier to infection exists in these plants. This assumption has prompted a number of experiments designed to remove such putative barriers and induce nodule-like structures on non-legumes. Earlier experiments had suggested that treatment of clover

187

188

Plantbiotechnology

t +

+

KDHnif \ 7 +~

n~i~ l~+nifB

TOther

nif/fixgenes

)

/

+

f/xN

Fig. 2. Organization and regulation of the nitrogen fixation genes. The genes are controlled by two transcriptional regulators, encoded by the and genes. These genes are, in turn, controlled by a two-component regulatory system consisting of a sensor component (FixL) and a transcriptional activator protein (FixJ). It is thought that FixL senses the concentration of oxygen which leads to selfphosphorylation, and this phosphate group is subsequently transferred to FixJ. Arrows denote the direction of transcription; (@), promoters; capital letters, gene products; (+), gene activation; (-), gene repression; (*), positions in the regulatory circuitry at which O 2 acts to modulate gene expression. Modified from [48"] and based on data from [49-51,52,,53.',54,55].

R. meliloti nifffix nifA fixK

f/xK

F-~

F FixJ(phosphorylated)

Hemoprotein

]

Microaerobiosis

Membrane

(nil~fix)

nif/fix

roots with cell walldegrading enzymes, such as cellu lase and pectolyase, could remove a barrier to rhizobial host-specificity [59]. This concept has now been e x tended to rice and oilseed rape which, when treated with a ceHulase-pectolyase enzyme mixture and infected with rhizobia in the presence of polyethylene glycol, formed bumps on the roots [60,61]. No nitrogen fixation was found in these structures, but limited microscopic studies suggest that rhizobia may be present intracellularly, although the plant cell cytoplasm appeared degraded [61].

Knoxville, Tennessee [62]. However, most of the above reports have not yet been scrutinized in detail or independently confirmed. Clearly, many more independent experiments need to be carried out to ascertain the significance of these observations.

In the absence of exogenously added cell wall-degrading enzymes, early events in the interaction of rhizobia with plants, such as root attachment and root hair curling, have been reported for monocot plants [62]. Rhizq bium attachment to root hairs of rice and oat seedlings has been demonstrated [63]. Engineered R trifoliistrains harboring a low-copy-number plasmid carrying the r o o t hair-curling genes (Hac, nod), have been reported to induce a root hair curling response in maize and rice plants [64]. Root hair curling in oats, induced by rhizobia has also been suggested [63,65]. Additionally, a non-nitrogen-fixing association of R astragaliwith barley roots has been described [66]. The induction of putative nodule-like structures on rice roots has been reported recently at the International Nitrogen Fixation Congress in

Few recent patents have been granted in the area of symbiotic nitrogen fixation research. R meliloti strains have been patented [P1.] which have been genetically engineered to overproduce the n/f4 gene products (Fig. 2), thus leading to improved (symbiotic) nitrogen fixation. The procedure of activating nodulation gene promoters of Rhizobium and related bacteria by using hybrid NodD (regulatory) proteins, encoded by chimeric nodD genes from different sources has also been patented [P2.], as well as the soybean Enod2 promoter [P3"].

Additional trends and patents in symbiotic nitrogen fixation research

A few additional approaches to improve symbiotic nitrogen fixation involving biotechnology have been summarized recently [4,5.]. In addition, the enhancement of symbiotic nitrogen fixation in alfalfa through the interaction of a toxin-producing strain of Pseudomonas

Biochemical and molecular studies of symbiotic nitrogen fixation de Bruijnand Downie 189

with nodulated plants has been suggested [67]. Increased growth, nodulation and nitrogen fixation and assimilation were correlated with selective alterations in the plant glutamine synthetase function, suggesting that manipulating the plant ammonium assimilation process may lead to improvement of symbiotic nitrogen fixation [67]. The isolation of a R meliloti mutant strain capable of effective nodulation of alfalfa plants in the presence of normally inhibiting concentrations of fixed nitrogen (ammonium) in the soil has been reported recently [68"]. A cytochrome mutant o f / ~ phaseoli with enhanced respiration and symbiotic nitrogen fixation has also been described [69]. Moreover, the recycling of hydrogen (a byproduct of nitrogenase catalysed reactions) by an uptakehydrogenase in bacteroids in the infected cells of soybean nodules, has been shown to increase plant productivity [70]. Various approaches to solve the Rbiz~ bium competition problem (i.e. the inability of superior inoculum strains to occupy nodules in soils containing large populations of indigenous rhizobia), and thus to increase symbiotic nitrogen fixation or to improve legume yield under controlled environmental conditions, have been reviewed recently [71"], however. Finally, and possibly of most importance from the point of view of an intermediate-term commercial payoff, is the work that has been carried out in an attempt to overcome the (negative) control of legume-nodulation [72..]. Soybean and pea mutants that form unusually high numbers of nodules even in the presence of normally inhibiting concentrations of nitrate have been isolated [72"']. These mutants may provide plant breeders with germplasm from which to develop more effective cultivars. The details of these approaches exceed the scope of this review.

Conclusions The world's rapidly expanding population clearly depends on plants for food, which in turn, depend on nitrogen compounds for their growth. Chemical fertilizers have been applied extensively in modem agriculture to replenish the rapidly depleting soil nitrogen sources, but this approach suffers from serious drawbacks such as high cost, accessibility problems and a severe negative environmental impact (nitrate contamination of the ground water [5°]), in both developed and developing regions of the world. Biological nitrogen fixation by symbiotic systems has been known for hundreds of years to be an agriculturally important and ecologically sound process to provide utilizable nitrogen to cultivated plants. In addition to upgrading existing technologies to improve both bacterial and plant partners, the use of biotechnology has been proposed vigorously as an important means to enhance (symbiotic) nitrogen fixation [4,5"]. However, relatively few concrete examples of biotechnologymediated breakthroughs in this area have been described during the 15 years or so that this type of research has been carried out. Primarily, this has been because of a severe underestimation of the complexity of the processes involved in nitrogen fixation and a lack of knowledge about their molecular basis. The studies reviewed here

on the rhizobial and plant genes responsible for nodulation and nitrogen fixation, their regulation and the signal transduction pathways involved, constitute an important step towards correcting this situation. It is to be expected that this progress, combined with the rapid advances in related areas of plant molecular biology summarized in other contributions in this issue, will result in a significant increase in useful applications, as well as a basic understanding of symbiotic nitrogen fixation.

Acknowledgements We would like to thank A Davis and K Pawlowski for preparing Figs 1 and 2, respectively, A Economou and S Rossbach for reading the manuscript and J Denarie, H Spaink, A Kondorosi, S Long, G Ditta, D Helinski and their collegues for ldndly maldng unpublished results available.

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.

GUTSCHmKVP: Energy Flows in the Nitrogen Cycle, Especially in Fixation. In Nitrogen Fixation Vol. 1 [book] edited by Newton WE, Orme-Johnson WH. Maryland: University Park Press, 1980, pp 17-27.

2.

LONG SR: Life Together In the Underground. Cell 1989, 56:203-214.

3. .

NAP JP, BISSELING T: Developmental Biology of a Plant Prokaryotic Symbiosis: The Legume Root Nodule. Science 1990, 250:948-954. A comprehensive review on plant and rhizobial genes involved in nodtile formation. 4.

McCORMICKD: How Biotechnology is Dealing with its Nitrogen Fixation. Biotechnology 1988, 6:383-385.

5. POSTGATEJ: Fixing the Nitrogen Fixers. New Scientist 1990, • 3:57-61. A historical perspective and discussion of the potential of biotechnology in biological nitrogen fixation. 6.

O'BRIANMR, MAIER RJ; Molecular Aspects of the Energetics of Nitrogen Fixation In Rhizobium-Legume Symbiosis. Biochem Biophys Acta 1989, 974:229-246.

7.

ARNOLDW, RUMP A, KLIPP W, PRIEFER UB, PUHLER A: Nucleotide Sequence o f a 24,206-bp DNA Fragment Carrying the Entire Nitrogen Fixation Gene Cluster of Klebsiella pneumoniae. J Mol Biol 1988, 203:715-738.

8. ..

LEROUGEP, ROCHE P, FAUCHER C, MAILLET F, TRUCHET G, PROMEJC, DENARIEJ: Symbiotic Host-specificity of Rhizo~ bium meliloti is Determined by a Sulphated and Acylated Glucosamine Oligosaccharide Signal. Nature 1990, 344:781-784. Using/~ melilot*'strains which overproduce symbiotic NOd factors, the major alfalfa-specific signal, NodRm-1, was purified by gel permeation, ion exchange and C18 reverse phase high-performance liquid chromatography. NodRm-1 was shown to be a sulphated tetrasaccharide of D-glucosamine, in which three amino groups were acetylated and one acylated with a C16 bis-unsaturated fatty acid. This compound was found to elicit root hair deformation on alfalfa, when added in nanomolar concentrations. A highly significant contribution to the field. 9. •

ROCHEP, LEROUGEP, PROMEJC, FAUCHERC, VASSEJ, MAR,LETF, GAMUTS, DE BILLYF, DENARIEJ, TRUCHET G: NodRm-1, a Sulphated Lipo-oligosaccharide Signal of Rhizobium meliloti,

190

Plant biotechnology Elicits Hair Deformation, Cortical Cell Division and Nodule Organogenesis o n Alfalfa Roots. In Molecular Genetics of Plant-Microbe Interactions [book] edited by Hennecke H, Verma DPS. Dordrecht, Boston, London: Kluwer Academic Publishers, 1991, p p 119-126. The purified, major symbiotic signal of/~ meliloti (NodRm-1) is shown to be able to induce all early stages of nodulation, including root hair curling, cortical cell divisions and nodule ontogeny. The nodH and nodQ genes are shown to be involved in attaching the sulphate moiety to NodRm-1. 10.

hal Molecules. In Molecular Genetics of Plant-Microbe Interactions [book] edited by Hennecke H, Verma DSP. Dordrecht, Boston, London: Kluwer Academic Publishers, 1991, pp 142-149. The R leguminosarum nodL gene product is shown to be capable of attaching an acetyl group to the reducing terminus of an acylated polyglucosamine molecule (equivalent to NodRm-1) produced by this strain, and this type of modification is postulated to introduce specificity into this class of signal molecule. 22.

BHUVANESWARI TV, SOLHEIMB: Root Hair Deformation in t h e W h i t e Clover-Rhizobium trifolii Symbiosis. Physiol Plant 1985, 63:25-34.

EAUCHER C, CAMUT S, DENARIE J, TRUCHET G: T h e nodH and nodQ Host Range G e n e s of Rhizobium meliloti Be. have as Avirulence Genes in R; leguminosarum bv. viciae and Determine C h a n g e s in t h e Production of Plant-specific Extracellular Signals. Mol Plant Microbe Interactions 1989, 2:291-300. The nodH and nodQ host genes o f / ~ meliloti are shown to be able to convert the vetch-specific signal produced by R leguminosarum by. viciae into an alfalfa-specific equivalent. 11. •

12. •.

SCHWEDOCKJ, LONG SR: ATP Sulphurylase Activity of the nodP and n o d Q Gene Products of Rhizobium meliloti. Nature 1990, 348:644-647. The R meliloti nodP and nodQ genes are shown to be functionally equivalent to the E coli cysCD gene products in being able to form adenosine 5'-phosphosulphate, which serves as a donor in sulphate transfer reactions, and therefore appear to be responsible for adding the sulphate moiety to NodRm-1 (see also [9o]). 13.

SHEARMANCA, ROSSEN L, JOHNSTON A'~(/B, DOWNIE JA: The Rhizobium G e n e nodF Encodes a Protein Similar to Acyl Carrier ProteIn and is Regulated by nodD Plus a Factor in Pea Root Exudate. EMBO J 1986, 5:647-652.

14.

BIBB MJ, BIRO S, MOTAMEDI H, COLLINSJF, HUCHINSON CR: Analysis of t h e Nucleotide Sequence of the Streptomyces glaucescens tmcl Gene Provides Information About the Enzymology of Polyketide Antibiotic Biosynthesis. EMBO J 1989, 8:2727-2736.

15.

SURINBP, DOWNIE JA: Rhizobium leguminosarum Genes Required for Expression and Transfer of Host-specific Nodulation. Plant Mol Biol 1989, 12:19-29.

SPAINKliP, WEINMANJ, DJORDJEVIC HA, WlJFFELMANCA, OKKER • RJH, LUGTENBERGBJJ: G e n e d c Analysis and Cellular Localization o f t h e Rhizobium Host-specificity-determining NodE Protein. EMBO J 1989, 8:2811-2818. The Rloizobium NodE protein is shown to be the major factor determining the host range of nodulation of R trifollii and 1~ leguminosarum and is found to be located in the cytoplasmic membrane.

23. •

ECONOMOUA, HAMILTONVdDO, JOHNSTON AWB, DOWNIE JA: The Rhizobium Nodulation Gene nodO Encodes a Ca 2+ Binding Protein that is Exported W i t h o u t N-Terminal Cleavage and is Homologous to Haemolysin and Related Proteins. EMBO J 1990, 9:349-354. The RIoizobium nodO gene is shown to encode an exported protein containing a domain homologous to repeated sequences in a group of bacterial exported proteins such as haemolysin. NodO is found to be capable of binding calcium and is proposed to interact directly with plant roots in a calcium-dependent manner, mediating an early stage in Rbizobium-plant recognition. DOWNIEJA, SUmN BP: Either of Two nod Gene Loci Can C o m p l e m e n t t h e Nodulation Defect of a nod Deletion Mutant of Rhizobium leguminosarum by. viciae. Mol Gen Genet 1990, 222:81-86. A deletion of R leguminosarum by. viciae lacking the host-specificity genes is found to be complementable by plasmids carrying the nodFE genes, as well as a plasmid carrying the nod(L)O gene(s), suggesting that nodulation can occur via two distinct pathways encoded by nonhomologous genes. 24. •

25.

DIAZ CL, MELEHJERS LS, HOOYKAAS PJJJ, LUGTENBERG BJJ, KIJNE JW: Root Lectin as Determinant of Host-Plant Specificity in t h e Rhizobium-Legume Symbiosis. Nature 1989, 338:579-581.

26.

VAN KAMMEN A: Suggested Nomenclature for Plant G e n e s Involved in Nodulation and Symbiosis. Plant Mol Biol Rep 1984, 2:43--45.

27.

VERMADPS, DELAUNEYAJ: Root Nodule Symbiosis: Nodulins and Nodulin Genes. In Plant Gene Research [book] edited by Verma DPS, Goldberg R. New York: Springer Verlag, 1988, pp 169-199.

16.

17.

SHELDONPS, KEKWICKRGO, SIDEBOTTOMC, SMITH CG, SLABAS APe 3-Oxoacyl-(acyl-carrier-protein) Reductase from Avocado (Persea americana) Fruit Mesocarp. Biochemistry 1990, 271:713-720.

18.

J O H N M, SCHMIDT J, WIENEKE U, KRtlSSMANN HI3, SCHELLJ: T r a n s m e m b r a n e Orientation and Receptor-like Structure of t h e Rhizobium meliloti C o m m o n Nodulation Protein NodC. EMBO J 1988, 7:583--588.

19.

SCHMIDTJ, WINGENDERR, JOHN M, WIENEKE U, SCHELLJ: Rhizobium meliloti nodA and nodB are Involved in Generating C o m p o u n d s that Stimulate Mitosis o f Plant Cells. Proc Natl Acad Sci USA 1988, 85:8578-8582.

20.

DOWNm JAz T h e nodL g e n e from Rhizobium leguminosarum is Homologous to t h e Acetyl Transferases Enc o d e d by lacA and cyeE. Mol Microbiol 1989, 3:1649-1651.

21. ••

SPAINKHP, GEIGER O, SHEELEY DM, VAN BRUSSELAAN, YORK WS, REINHOLD xgrN, LUGTENBERGBJJ, KENNEDY EP: T h e Biochemical Function of Rhizobium leguminosarum Pro. teins Involved in t h e Production of Host-specific K Sig-

DE MAAGD RA, SPAINK HP, PEES E, MULDERS IHM, WIJFJES A, WIJFFELMAN CA, OKKER RJH, LUGTENBERG BJJ: Localization and Symbiotic Function of a Region on t h e Rhizobium leguminosarum Sym Plasmid Responsible for a Secreted Flavonoid-inducible 5 0 k D Protein. J Bacteriol 1989, 171:1151-1157.

28.

SCHERESB, VAN ENGELEN F, VAN DER KNAPPE E, VAN DE WIEL C, vAN KAMMENA, BISSELINGT: Sequential Induction of Nodulin Gene Expression in t h e Developing Pea Nodule. Plant Cell 1990, 2:687-700. A set of cDNA clones representing early nodulin mRNAs from pea nodules are characterized and their temporal and spatial expression patterns in the developing nodule are investigated using in situ hybridization techniques. •

29. ••

SCHERESB, VAN DE WIEL C, VAN ECK H, ZWARTKRtnSF, WOLTERS AM, GLOUDEMANST, VAN KAMMENA, BISSELINGT: T h e E n o d l 2 G e n e Product is Involved in t h e Infection Process DurIng the Pea-Rhizobium leguminosarum Interactions. Cell 1990, 60:281-294. The early nodulln Enodl2 of pea is postulated to be involved in the infection process, as the expression of the Enod12 gene is found in cells containing infection threads and those shortly ahead of them. Rhizc> bium c o m m o n and host-specific nodulation gene products are shown to be essential for Enod12 expression. Differential Enod12 expression is found in nodules and flowers of the infected plants. 30. ••

vAN DE WIEL C, SCHERES B, FRANSSENH, VAN LIEROP i J , VAN LAMMERENA, VAN KAMMENA, BISSELING T: T h e Early Nodulin Transcript Enod2 is Located in t h e Nodule P a r e n c h y m a (Inner Cortex) of Pea and Soybean Root Nodules. EMBO J 1990, 9:1-7.

Biochemical and molecular studies of symbiotic nitrogen fixation de Bruijn and Downie The expression of the early nodulin Enod2genes of pea and soybean is found to be confined to the inner cortex (nodule parenchyma) of the respective nodules. A possible role for the Enod2 protein in creating an oxygen barrier for the central Rhizobium-infected tissue is postulated.

Spatial Patterns of Expression in Transgenic Lotus cornicu l a t u s Plants. Plant Cell 1989, 1:391-401.

DE BRUIN FJ, SZABADOS L, SCHELLJ: Chimeric Genes and Transgenic Plants are Used to Study the Regulation of Genes Involved in Plant-Microbe Interactions (Nodulin Genes). Dev Genet 1990, 11:182-196. The role of c/~acting elements and transacting elements in nodulespecific expression o f nodulin genes is reviewed in detail.

FORDEBG, FREEMANJ, OLIVERJE, PINADAM: Nuclear Factors Interact with Conserved A/T-Rich Elements Upstream of a Nodule,enhanced Glutamine Synthetase Gene from French Bean. Plant Cell 1990, 2:925-939. The 5'-upstream region of the nodule-enhanced glng gene of French bean is found to have conserved motifs involved in nodule-enhanced expression and binding of nuclear trans~acting factors (see also [42,43]).

32.

DICKSTEINRT, BISSELING T, REINHOLD VN, AUSUBEL FM: Expression o f Nodule-specific genes in Alfalfa Root Nodules Blocked at an Early Stage o f Infection. Genes Dev 1988, 2:677-678.

42.

JENSENEO, MARCKERKA0 SCHELLJ, DE BRUIJN VJ: Interaction of a Nodule-specific Trans-acting factor with Distinct DNA Elements in the Soybean Leghemoglobin lbc3 5'-Upstream Region. EMBO J 1988, 7:1265-1271.

VAN DE WIEL C, NORRIS JH, BOCHENEK B, DICKSTEIN R, BISSELINGT, HIRSCH AM: Nodulin Gene Expression and Enod2 Localization In Effective, Nitrogen-fixing and Ineffective, Bacteria-free Nodules of Alfalfa. Plant Cell 1990, 2:1009-1017. On the basis of the spatial patterns of the early nodulin Enod2 gene expression, the developmental pathway responsible for the generation of bacteria-free nodules (bacterially or chemically induced) is found to be the same as that for nitrogen-fixing nodules. Auxin transport inhibitors are reported to mimic factors normally triggering nodule development.

43.

METZ BA, WELTERS P, HOFFMANN HJ, JENSEN EO, SCHELLJ, DE BRU1JN FJ: Primary Structure and Promoter Analysis of Leghemoglobin Genes of the Stem-nodulated Tropical Legume Sesbania rostratae Conserved Coding Sequences, Cis-elements and Trans-acting Factors. Mol Gen Genet 1988, 214:181-191.

31. •

33.

41. •

•

34.

HIRSCHAM, BUWANESWARITV, TORP,EY JG, BISSELINGT: Early Nodulin Genes are Induced in Alfalfa Outgrowths Elicited by Auxin Transport Inhibitors. Proc Natl Acad Sci USA 1989, 86:1244-1249.

44.

JACOBSENK, LAURIDSONNB, JENSEN EO, MARCKERA, POULSON C, MARCKER K& HMG-l-like Proteins from Leaf and Nodtile Nuclei Extracts Interact with Different AT Motifs in Soybean Nodulin Promoters. Plant Cell 1990, 2:85-94. One of the nuclear DNA-binding proteins found to interact with an A/Trich element in the soybean leghemoglobin/bc3 5'-upstream region is shown to have properties in common with mammalian high mobility group-type proteins. •

TRUCHETG, BARKERnG, CAMUTS, DE BILLyF, MASSEJ, HUGUET T: Alfalfa Nodulation in the Absence of Rhizobiun~ Mol Gen Genet 1989, 219:65-68. Under axenic conditions, certain alfalfa plants are shown to develop non-nitrogen-fixing structures on their roots which histologically closely resemble normal nodules. The early nodulin gene Enod2 is reported to be expressed in these nodules. Thus, Rhizobium is not absolutely required for nodule ontogeny.

45.

HILL S: HOW is Nitrogenase Regulated by Oxygen? FEMS Microbiol Lett 1988, 54:111-130.

46.

APPLEBYCA: Leghemoglobin and R h i z o b i u m Respiration. A n n u Rev Plant Physiol 1984, 35:443-478.

47.

ALBRIGHTLM, HUALAE, AUSUBELFM: Prokaryotic Signal Transduction Mediated by Sensor and Regulator Protein Pairs. A n n u Rev Genet 1989, 23:311-336.

36.

48.

35. •

37.

LONGSR, COOPERJ: Overview of Symbiosis. in Molecular Ge~ netics of Plant--Microbe Interactions [book] edited by Palacios 1g Verma DPS. Minnesota: APS Press, 1988, pp 163-178. DE BRUIJN FJ: The Unusual Symbiosis Between the Diazotrophic Stem-nodulating Bacterium A z o r h i z o b i u m c a u l i n o d a n s ORS571 and Its Host, the Tropical Legume S e s b a n i a rostrata. In Plant Microbe Interactions Vol 3 [book] edited by Nester EW, Kosuge T. New York: McGrawHill, 1989, pp 457-504.

38. ••

STOUGAARD J, JOERGENSON JE, CHRISTENSEN T, KUHLE A, MARCKERK& Interdependence and Nodule Specificity of Ci&acting Regulatory Elements in the Soybean Leghemoglobin /bc3 and N23 Gene Promoters. Mol Gen Genet 1990, 220:353-360. By analysing the expression of chimeric soybean leghemoglobIn ~c3-chloramphenicol acetyitransferase gene fusions in transgenic Lotus plants, c~acting elements responsible for nodule-specific expression are delimited. An interdependence of an upstream strong positive element and an organ (nodule) specificity element for efficient nodulespecific expression is demonstrated. SZABADOSIo RATET P, GRUNENBERG B, DE BRUIJN FJ: Functional Analysis of the Sesbania rostrata Leghemoglobin glb3 Gene 5' Upstream Region in Transgenic Lotus corn i c u l a t u s and N i c o t i a n a t a b a c u m Plants. Plant Cell 1990, 2:973-986. Chimeric Sesbania rostrata leghemoglobin g/b3-[]-glucuronidase (GUS) gene fusions are used to characterize c~-acting elements responsible for general transcriptional control and nodule-specific expression. A histological GUS staining protocol is used to show that infected cell-specific /b gene expression In nodules is mediated by its 5'-upstream region.

DE BRUIJNFJ, HILGERTU, STIGTERJ, SCHNEIDERM, MEYERZAH, KLOSSE U, PAWLOWSKI K: Regulation of Nitrogen Fixation and Assimilation Genes in the Free-living Versus Symbiotic State. In Nitrogen Fixation: Achievements a n d Objectives [book] edited by Gresshoff PM, Roth LE, Stacey G, Newton WE. New York, London: Chapman and Hall, 1990, pp 33-44. This review compares the regulation Of nitrogen fixation and assimilation genes of the free-living diazotroph K peumoniae, the symbiotic nitrogen fixing bacteria/~ meliloti and B. japonicum and the facultative diazotroph/symbiotic nitrogen-fixing bacterium A caulinodans. •

49.

VIRTSEL, STANFIELD SW, HELINSKI DR, DITrA GS: Common Regulatory Elements Control Symbiotic and Microaerobic Induction of nifA in R h i z o b i u m meliloti. Proc Natl Acad Sci USA 1988, 85:3062-3065.

50.

DAVIDM, DAVERANML, BATUT J, DEDIEU A, DOMERGUE O, GHAI J, HERTIG C, BOISTARDP, KAHN D: Cascade Regulation of n i l Gene Expression in R h i z o b i u m meliloti. Cell 1988, 54:671-683.

51.

BATUTJ, DAVERAN-MINGOTML, DAVID M, JACOBSJ, GARNERONE AM, KAHN D: F/x/( a Gene Homologous with f n r and crp from Escherichia coli, Regulates Nitrogen Fixation Genes both Positively and Negatively in R h i z o b i u m melilotz: EMBO J 1989, 8:1279-1286.

39. ••

40.

FORDE BG, DAY HM, TURTON JF, WEN-JUN S, CULLIMORE J, OLIVER JE: Two Glutamate Synthetase Genes from Phaseolus vulgaris L. Display Contrasting Developmental and

52. •

DE PHILIP P, BATUTJ, BOISTARD P: R h i z o b i u m meliloti F/XL is an Oxygen Sensor and Regulates R. meliloti nifA and f i x K Genes Differently in Escherichia coli. J Bacteriol 1990, 172:4255-4262. Nifli-lac and fixK-lac gene fusions and plasmids carrying the fixLJ genes are used to show that the /~ meliloti fixJ product senses oxygen in the heterologous host E. coli. This is consistent with the fixLJmediated microaerobic induction of n ~ and fixK in /~ meliloti. It is also shown that tufA and fixKpromoters are differentially activated by FixJ in response to the oxygen signal.

191

192

Plantbiotechnology 53. o•

GILLIs-GONZALEZMA, DITI'A GS, HELINSKIDR: A Haemoprotein with Kinase Activity Encoded by the Oxygen Sensor of Rhizobium meliloti. Nature 1991, 150:170--172. The ~ meliloti nitrogen fixation regulatory gene fixL is shown to encode an •x-binding haemoprotein with kinase activity, that is capable of autophosphorylation and transfer of the phosphate group to its partner (thereby activating it), the transcriptional regulator FixJ, in response to microaerobic conditions. This is a major advance in the elucidation of the signal transduction pathways involved in controlling rhizobial niJ~fix gene expression. 54.

55.

BEYNONJL, WILLIAMSMK, CANNON FC: Expression and Functional Analysis o f the Rhizobium meliioti nifA Gene. EMBO J 1988, 7:7-14. FISCHERH i , BRUDERERT, HENNECKE H: Essential and Nonessential Domains in the Bradyrhizobium japonicum NifA Protein: Identification of Indispensable Cysteine Residues Potentially Involved in Redox Reactivity and/or Metal Binding. Nucleic Acids Res 1988, 16:2207-2224.

56. HENNECKEH: Regulation of Bacterial Gene Expression by • Metal-protein Complexes. Mol Microbiol 1990, 4:1621 1628. In this review, the role of metal ions as essential cofactors in several transacting bacterial regulatory proteins is explored. Speculations are presented about the relationship between metal-dependence of the rhizobial NifA and E. coliFnr proteins and their regulatory role in respondhag to oxygen. 57.

TRINICKMJ, GALBRA1THJ: The Rhizobium Requirements of the Non-legume Parasponia in Relationship to the Crossinocculation Group Concept o f Legumes. New Phytol 1980, 86:17-26.

58.

LALONDEM, SIMON L, BOUSQUETJ, SEGUINA: Advances in the Taxonomy of Frankia: Recognition of Species alni and elaeagni and Novel Subspecies pommerii and vandykii. In Nitrogen Fixation: Hundred Years After [book] edited by Bothe H, de Bruijn FJ, Newton WE. Stuttgart, New York: Fischer Verlag, 1988, pp 671-680.

59.

AL-MALLAH MK, DAVEY MR, COCKING EC: Emzymatic Treatment of Clover Root Hairs Removes a Barrier to Rhizobium-host Specificity. Biotechnology 1987, 5:1319-1322.

cient Nodulation Capacity on Alfalfa in the Presence of Ammonium. Mol Gen Genet 1989, 219:89-96. Expression of the ~ meliloti nodulation (nod) genes is shown to be affected by the central nitrogen regulation (ntr) system. A/~ meliloti mutant is described with an altered sensitivity to ammonia regulation, capable of more efficient nodulation of alfalfa in the presence of ammonia. 69.

SOBERONM, WILLIAMSHD, POOLE ILK, ESCAMILLAE: Isolation of a Rhizobium phaseoli Cytochrome Mutant with Enhanced Respiration and Symbiotic Nitrogen Fixation. J Bacteriol 1989, 171:465-472.

70.

EVANSHJ, RUSSELLSA, HANUS VJ, PAPEN H, SAYAVEDRASOT• L, ZUBER M, BOURSmR P: Hydrogenase and Nitrogenase Relationships in Rhizobiurr~ Some Recent Developments In Nitrogen Fixation: Hundred Years After [book] edited by Bothe H, de Bruijn FJ, Newton WE. Stuttgart and New York: Fischer Verlag, 1988, pp 577-582.

71. •

TRIPLETrEW: The Molecular Genetics of Nodulation Cornpetitiveness in Rhizobium and Bradyrhizobiurt~ Mol PlantMicrobe Int 1990, 3:199-206. In this review, many important parameters of nodulation competitiveness are summarized and approaches to improve rhizobial competitiveness are discussed. 72. e•

CARROLLBJ, MATHEWSA: Nitrate Inhibition of Nodulation in Legumes. In Molecular Biology of Symbiotic Nitrogen Fixation [book] edited by Gresshoff P. Florida, USA: CRC Press, 1990, pp159-180. Reviews present knowledge about nitrate inhibition of nodule formation on legume roots, as well as characterizing nitrate-tolerant, supernodulating mutants.

Annotated patents • 0o

of interest of outstanding interest

60.

AL-MAILAHMK, DAVEYMR, COCKINGEC: Formation of Nodular Structures on Rice Seedlings by Rhizobia. J Exp Bot 1989, 40:473-478.

61.

AL-MALLAHMK, DAVEY MR, COCKING EC: Nodulation of Oilseed Rape (Brassica napus) by Rhizobia. J Exp Bot 1990, 41:1567-1572.

BIOTECHNICAINTERNATIONALINC: Improved Biological Nitrogen Fixation using Microorganisms Transformed with Vectors Containing Genes for Increasing Conversion of Nitrogen to Ammonia. 14/4/88 88US-181430. 2/11/89 EP-339830 A. g meliloti strains harboring gene constructs leading to the overproduction of the nitrogen fixation-specific regulatory g e n e m f , t are constructed and found to lead to increased levels of nitrogen fixation and alfalfa plant growth in the field.

62.

SIMONMOFFAT A: Nitrogen-fixing Bacteria Find N e w Partnet's. Science 1990, 250:910-913.

P2. •

63.

TEROUCHIN, SYONO K: Rhizobium Attachment and Curling In Asparagus, Rice and Oat Plants. Plant Cell Pbysiol 1990, 31:119-127.

64.

PLAZINSmJ, INNES RW, ROLFE BG: Expression of Rhizobium trifolii Early Nodulation Genes on Maize and Rice Plants. J Bacteriol 1985, 163:812-815.

65.

TEROUCHIN, SYONO K: Hair Curling Induced in Heterologous Legumes and Monocots by Flavonoid-treated Rhizobia. Plant Cell Physiol 1990, 31:113-118.

66.

JING Y, ZHANG BT, SHAN XQ: Pseudonodule Formation on Barley Roots Induced by Rhizobium astragali. FEMS Microbiol Lett 1990, 69:123-128.

67.

KNIGHTTJ, LANGSTON-UNKEFERPJ: Enhancement of Symbiotic Nitrogen-fixation by a Toxin-releasing Plant Pathogen. Science 1988, 241:951--954.

68. •

DUSHAI, BAKOS A, KONDOROSI A, DE BRUIJN FJ, SCHELLJ: The Rhizobium meUloti Early Nodulation Genes (nodABC) are Nitrogen-regulated: Isolation o f a Mutant Strain with Eft]-

P]. .

ROKSUNIVERSrrEITLEIDEN:Activating Nodulation Promoters o f Rhizobium and Related Bacteria using Hybrid nodD Gene Obtained by Recombination Between nodD Genes. 16/9/88 88NL-00294. 22/3/90 WO9002805. Chimeric rhizobial NodD proteins are constructed, which lead to inducible nod gene ( n o d promoter) expression in response to a wider range of plant-derived inducing compounds and/or at a higher level. P3. •

LUBRIZOLGENETICS: Enod2 Gene Regulatory Region used for Early Expression of Structural Gene in Developing Root Nodule o f Soybean Plant. 1/7/88 88US-214297. 3/1/90 EP349338. The DNA sequence of the soybean Enod2 gene promoter is determined. It is proposed that the promoter directs the specific expression of chimeric genes in developing nodules.

FJ de Bmijn, MSU-DOE Plant Research Laboratory and Department of Microbiology, Michigan State University, East Lansing, Michigan 48824, USA. JA Downie, John Innes Institute, AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK,