Journal of Heredity 2003:94(2)
© 2003 The American Genetic Association 94:191-193
Brief Communication |
Allelic Relationships of Pea Nodulation Mutants
From the Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde
ská l083 Prague, CZ-142 20 Czech Republic.
Address correspondence to K. Novák at the address above, or e-mail: novak{at}biomed.cas.cz.
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Abstract |
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Thirteen stable nonnodulating mutant lines of pea (Pisum sativum L.) originating from cv. Finale were tested for allelism in pairwise crosses. The F1 plants were evaluated for the symbiotic phenotype under controlled growth conditions against the nodule bacterium Rhizobium leguminosarum bv. viciae strain 248. All mutations were found to be recessive and the lines were classified into eight complementation groups comprising Risnod1-Risnod23, Risnod8, Risnod9-Risnod22, Risnod14, Risnod19-Risnod25, Risnod20, Risnod24-Risnod26, and Risnod32. Position of Risnod21 was not firmly established, leaving the possibility of allelism both with Risnod19-Risnod25 and Risnod20. The results were partially consistent with the previous reports on the allelism of these lines. Additional crosses confirmed the correspondence of Risnod14 with the locus sym7 and of Risnod19-Risnod25 with sym8. The high number of eight complementation groups formed by 13 mutants provides an indication of additional nodulation loci in pea to those already reported and confirms the complexity of the genetic control of the early stages of nodulation.
Pea (Pisum sativum L.) remains one of the most important host plant models for studying nodule bacteria symbiosis in leguminous plants. This economically important feature of legumes, providing nitrogen autotrophy by bacterial fixation of atmospheric nitrogen, has attracted increasing attention during recent years when the efforts of many rhizobial symbiosis laboratories shifted to the host plant (e.g., Downie and Walker 1999; Provorov et al. 2002). One of the traditional approaches to deciphering the symbiosis consists in the genetic dissection of the process, using a set of induced symbiotic mutants (Wais et al. 2000; Walker et al. 2000). In spite of easier and more efficient mutagenesis in model legumes such as Lotus japonicus and Medicago truncatula due to their small genomes, small plant size, and a faster generation time (Wais et al. 2000), pea is still comparable with respect to the number of symbiotic mutants available. Most of them have been accumulated during the last two decades (e.g., Borisov et al. 1993; Duc and Messager 1989; Engvild 1987; Jacobsen 1984; Kneen et al. 1994). The wealth of mutant pea lines has not been completely classified into complementation groups. Therefore, the number of pea genes involved in symbiosis establishment that can be mutationally revealed is still not final.
To date, 41 distinct loci have been identified, and most of them numbered from sym-1 to sym-40, except for those traditionally denoted as nod1, nod2, and nod3 (Provorov et al. 2002). Among them, mutations in only 12 loci (sym7, sym8, sym9, sym10, sym11, sym14, sym19, sym30, sym34, sym35, sym36, and sym38) are supposed to lead to the nonnodulation phenotype, while the other control nodule effectiveness and nodule number (Provorov et al. 2002). Consequently the products of such genes can be considered as involved in the symbiosis initiation. To learn about the complexity of nodulation control in a set of partially classified nodulation mutants obtained by Dr. K. Engvild (Engvild 1987; at Risø National Laboratory, Denmark), crosses were carried out among 13 nonnodulating (Nod–) lines from this group to determine recessiveness/dominance of the mutations and their mutual complementarity.
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Materials and Methods |
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Plant Materials
The pea lines involved were generated with ethyl methane sulfonate mutagenesis in garden pea (Pisum sativum L.) cv. Finale (Engvild 1987) (kindly supplied by Dr. K. Engvild). The lines have been propagated at the Institute of Microbiology, Prague, since 1988. Although 32 lines in the original collection bear the notation Risnod, meaning their original description as Nod– mutants, only 13 lines were included in the present study for the following reasons. Line Risnod18 has not been successfully propagated at the Risø National Laboratory (Engvild K, personal communication). Another six lines (Risnod6, 7, 11, 13, 16, and 28) were lost or not propagated at the Institute of Microbiology because of their low viability. Eight lines were found to be nodulating in a growth chamber test (Novák et al. 1993): Risnod9 and Risnod26 formed tiny white nodules, being blocked after nodule initiation, Risnod3, 4, 5, and 17 formed inefficient nodules of different appearance, while Risnod15, 29, and 31 formed efficient nodules, matching the class of conditionally nodulating mutants. Five more mutants (Risnod2, 10, 12, 27, and 30) exhibited an unstable symbiotic phenotype or decreased viability (Novák K, unpublished data). Their crosses were therefore substantially delayed. In addition, pea lines JII3027 (E69) and JII3028 (R25), carrying symbiotic mutations in loci sym7 and sym8, respectively, were obtained from the John Innes Pisum Collection (John Innes Center, Norwich, UK) and were crossed with the 13 lines above.
Crosses and Progeny Analysis
Field-grown plants were crossed in the seasons 1995–2001. F1 plants were analyzed for symbiotic phenotypes under controlled conditions in view of the frequently observed phenotypic instability of the nodulation mutants. The seeds were surface sterilized with a 2% solution of chloramine B for 40 min and sown without rinsing 3 cm deep into autoclaved polypropylene troughs containing coarse expanded clay pellets (fraction 4–8 mm) and watered with nitrogen-free nutrient solution according to 
krdleta et al. (1980). The troughs were covered with sterile aluminum foil to prevent contamination with rhizobia and pathogens. Forty-eight hours after sowing, the troughs were inoculated with 40 ml of bacterial suspension containing 107 cell/ml of the wild-type strain Rhizobium leguminosarum bv. viciae 248 (Josey et al. 1979). The suspension had been prepared in distilled H2O by scraping bacterial colonies off the surface of yeast extract-mannitol agar plates (Vincent 1970) after 4 days of incubation at 28°C. After pea seed germination at 18°C, the cover was removed and the plants were grown under 500 µE of photosynthetically active radiation at 22°C/16°C and a 16 h day/8 h night regime in a growth chamber (Conviron S10H). The nutrient solution was changed twice a week until root phenotype evaluation.
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Results and Discussion |
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The first nodules appeared 2 weeks after inoculation, while pink efficient nodules developed 3 weeks after inoculation, at the six-leaf stage (including two rudimentary leaves). The root phenotype was estimated 4 weeks after inoculation, when the nonfixing plants already exhibited symptoms of nitrogen deficiency in the shoot (yellowing of lower leaves and slowed growth). The stability of the nonnodulation phenotype of the parental lines was confirmed by sowing them as a negative control with F1 hybrids. On the other hand, the parental cultivar Finale served as a positive, nodulating control to distinguish deviations in the F1 nodulation from the wild type.
Since no substantial differences were observed in reciprocal crosses, which were done in 36 of 64 combinations, the results of direct and reciprocal crosses were pooled. Phenotypes of at least three F1 plants were analyzed in each combination. After establishing allelism between a pair of mutants, the results of crosses of these two mutants with other complementation groups were pooled (Table 1).
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All 13 mutants were recessive for nonnodulation in crosses onto the parental cultivar Finale. The mutants were classified into eight complementation groups, as indicated in Table 1. A comparatively high number of complementation groups was found in a limited set of mutants, suggesting that many loci are involved in the early stages of nodulation. On the other hand, the reduction in the number of mutants to the number of complementation groups is helpful in relating them to the already established mutant loci (Borisov et al. 1999; Brewin et al. 1993; Kneen et al. 1994). The recessiveness of the mutations and the allelism of Risnod9 with Risnod22 and Risnod24 with Risnod26 correspond to data obtained by Dr. K. Engvild in 1988 (Engvild K, personal communication). The observed allelism in the pair Risnod19-Risnod25 corresponded to data presented by Gianinazzi-Pearson et al. (1991), while Risnod1-Risnod23, Risnod9-Risnod22, and Risnod24-Risnod26 pairs were consistent with data from Dr. G. Duc and Dr. M. Sagan, as reported by Borisov et al. (1999). Risnod32 seems to form another complementation group. However, relation of this group to the previously established sym loci has not been completed. Allelism between the Risnod19-Risnod25 group and Risnod21 (Borisov et al. 1999; Engvild K, personal communication) was not confirmed in the present work, and contradictory data suggest allelism with Risnod20. For this reason, Risnod21 is not included in Table 1. The only maternal plant effect observed was a frequent formation of pale and less-efficient nodules when allelic lines Risnod24 and Risnod26 served as the maternal plant.
The crosses with lines JII3027 and JII3028 confirmed the correspondence of the established complementation group of Risnod14 with the locus sym7 (Borisov et al. 1999) and preliminarily confirm the correspondence of Risnod19-Risnod25 with sym8. Crosses of other Risnod line groups with the two above lines confirmed complementarity with sym7 and sym8, although the number of F1 analyzed is still not final.
The main implication that can be drawn is about the complexity of the early stages of nodulation. The eight loci controlling nodulation itself, as revealed in this limited set of 12 mutants derived from one cultivar (Table 1), is close to the 12 loci established mainly with use of mutant sets originating from cultivars Sparkle, Frisson, Rondo, SGE, Sprint-2, and Ramonsky-77 (Borisov et al. 1999). Taking into account the number of double-hit genes and the binomial distribution of mutations, the most probable number of pea genes governing nodule initiation which might be detected by mutagenesis is around 13, provided that their mutation rate is equal. This figure corresponds to the number of loci reported to date (Borisov et al. 1999; Provorov et al. 2002), however, additional genes involved in the root interaction with rhizobial nodulation factors and in signal transduction (Spaink 2000) are not excluded.
The lines Risnod20, 22, and 25 are blocked very early in the nodulation process, as judged partly from microscopic data. On the other hand, sym7 and sym8 code for products acting very early in nodulation, as shown by Walker et al. (2000). This implies that Risnod14, 20, 22, and 25 might correspond to the four genes controlling the earliest stages of symbiotic interactions in a model legume Medicago truncatula (genes DMI1, DMI2, DMI3, and NSP), as resolved by Catoira et al. (2000).
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Acknowledgments |
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This work was supported by grant 521/00/0937 of the Grant Agency of the Czech Republic and by the institutional research concept (no. A V0Z5020903).
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Footnotes |
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Corresponding Editor: Prem P. Jauhar
Received May 30, 2002
Accepted December 31, 2002
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References |
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Borisov AY, Jacobi LM, Lebsky VK, Morzhina EV, Tsyganov VE, Voroshilova VA, Tikhonovich IA, 1999. Genetic system controlling development of nitrogen-fixing nodules and arbuscular mycorrhiza. Pisum Genetics. 31:40-43.
Borisov AY, Morzhina EV, Kulikova OA, Tchetkova SA, Lebsky VK, Tikhonovich IA, 1993. New symbiotic mutants of pea (Pisum sativum L.) affecting either nodule initiation or symbiosome development. Symbiosis. 14:297-313.
Brewin NJ, Ambrose MJ, Downie JA, 1993. Root nodules, Rhizobium and nitrogen fixation. In: Peas: genetics, molecular biology and biotechnology (Casey R and Davies DR, eds). Wallingford: CAB International; 237–290.
Catoira R, Galera C, De Billy F, Penmetsa RV, Journet E-P, Maillet F, Rosenberg C, Cook D, Gough C, Dénarié J, 2000. Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway. Plant Cell. 12:1647-1665.
Downie JA, Walker SA, 1999. Plant responses to nodulation factors. Curr Opin Plant Biol. 2:483-489.[CrossRef][Web of Science][Medline]
Duc G, Messager A, 1989. Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation. Plant Sci. 60:207-213.[CrossRef]
Engvild KC, 1987. Nodulation and nitrogen fixation mutants of pea, Pisum sativum. Theor Appl Genet. 74:711-713.
Gianinazzi-Pearson V, Gianinazzi S, Guillemin JP, Trouvelot A, Duc G, 1991. Genetic and cellular analysis of resistance to vesicular arbuscular (VA) mycorrhizal fungi in pea mutants. In: Advances in molecular genetics of plant-microbe interactions, vol. 1 (Hennecke H and Verma DPS, eds). Dordrecht: Kluwer Academic; 336–342.
Jacobsen E, 1984. Modification of symbiotic interaction of pea (Pisum sativum L.) and Rhizobium leguminosarum by induced mutations. Plant Soil. 82:427-438.[CrossRef]
Josey DP, Beynon JL, Johnston AWB, Beringer J, 1979. Strain identification in Rhizobium using intrinsic antibiotic resistance. J Appl Microbiol. 46:343-350.
Kneen BE, Weeden NF, LaRue TA, 1994. Non-nodulating mutants of Pisum sativum (L) cv. Sparkle. J Hered. 85:129-133.
Novák K, 
krdleta V, N
mcová M, Lisá L, 1993. Symbiotic traits, growth, and classification of pea nodulation mutants. Rostlinná V
roba (Prague). 39:157-170.
Provorov NA, Borisov AY, Tikhonovich IA, 2002. Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza. J Theor Biol. 214:215-232.[CrossRef][Web of Science][Medline]
krdleta V, Gaudinová A, Nmcová M, Hyndráková A, 1980. Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulated Pisum sativum L. Folia Microbiol. 25:155-161.
Spaink HP, 2000. Root nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol. 54:257-288.[CrossRef][Web of Science][Medline]
Vincent JM, 1970. A manual for the practical study of root-nodule bacteria. Oxford: Blackwell Scientific.
Wais RJ, Galera C, Oldroyd G, Catoira R, Penmetsa RV, Cook D, Gough C, Dénarié J, Long SR, 2000. Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. Proc Natl Acad Sci USA. 97:13407-13412.
Walker SA, Viprey V, Downie JA, 2000. Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by Nod factors and chitin oligomers. Proc Natl Acad Sci USA. 97:13413-13418.
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