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A Co-opted Regulator of Lateral Root Development Controls Nodule Organogenesis in Lotus
Developmental Cell
Previews A Co-opted Regulator of Lateral Root Development Controls Nodule Organogenesis in Lotus Rachel Shahan1 and Philip N. Benfey1,* 1Duke University and Howard Hughes Medical Institute, Durham, NC 27701, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.12.009
Legumes, a subset of flowering plants, form root nodules in symbiosis with nitrogen-fixing bacteria. The regulatory network controlling nodule formation has remained mysterious. In a recent issue of Science, Soyano et al. (2019) demonstrate that co-option of an existing lateral root developmental program is used in Lotus for nodule organogenesis. Foundational questions in developmental biology concern the mechanisms underlying organ initiation and patterning. Postembryonic organogenesis is a feature unique to plants and is mediated by maintenance of stem cell populations in specialized niches. Two such niches, the primary shoot and root apical meristems, are formed in the developing embryo and are responsible for growth of the primary shoot and root, respectively. However, the post-embryonic formation of lateral organs is not directly governed by divisions of the primary meristems. Lateral roots are formed by divisions of specific, differentiated pericycle cells in response to environmental conditions (Malamy and Benfey, 1997). Intriguingly, the roots of several orders of flowering plants, most notably the legumes, are capable of forming an additional lateral organ, the nodule. Initiated by symbiotic interaction with nitrogen-fixing bacteria, legume nodules arise from divisions of differentiated cortical cells (Hirsch et al., 1997). Although lateral roots and nodules are both derived from primary root tissues, the evolutionary origin of the nodule and the molecular regulation of its organogenesis have long been mysterious. In a recent issue of Science, Soyano et al. (2019) show that a shared master regulator controls both lateral root and nodule organogenesis in Lotus japonicus, thereby providing evidence for co-option of an existing regulatory network for the development of a unique organ in legumes. Although all eudicots produce lateral roots, only a subset of genera within a single clade produce nodules and engage in symbiotic relationships with nitrogenfixing bacteria, collectively called rhizobia (Griesmann et al., 2018). Atmospheric ni-
trogen is abundant but unusable, while mineral forms are limiting for plant growth. Legumes such as pea, clover, and soy form nodules in order to host rhizobia in optimal conditions for nitrogen fixation. The host plant provides colonized rhizobia with a carbon source and, in exchange, benefits from the reduction of atmospheric nitrogen into ammonium. In this way, legumes are able to thrive in low-nitrogen terrestrial environments. Induction of nodulation in non-leguminous species could reduce nitrate fertilizer usage and revolutionize agriculture around the globe, but this goal first requires detailed knowledge of the molecular regulation of nodule organogenesis. Genetic approaches have identified several key regulators of nodule formation in legumes. One such nodulation-specific factor is Lotus japonicus NODULE INCEPTION (NIN), a transcription factor required for rhizobia infection and nodule organogenesis (Schauser et al., 1999; Marsh et al., 2007). NIN activates two genes that encode different subunits of a Nuclear Factor-Y (NF-Y)-binding protein complex. Ectopic expression of NIN and its two targets causes abnormal cell division during lateral root development and activates cortical cell division during nodule organogenesis, suggesting a link between nodule and lateral root developmental programs (Soyano et al., 2013). However, an enduring question remains: do nodule and lateral root organogenesis share a common regulatory pathway? Differences in response to cytokinin initially hinted that the answer is no. Cytokinin signaling is necessary and sufficient to induce nodule formation through induction of NIN but suppresses lateral root development (Bensmihen, 2015).
6 Developmental Cell 52, January 6, 2020 ª 2019 Elsevier Inc.
To dissect the molecular pathway underlying legume organogenesis, Soyano et al. (2019) examined existing ChIP-seq data to identify NIN target candidates with induced transcription in response to rhizobia. Only one candidate gene, ASYMMETRIC LEAVES 2-LIKE 18/ LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/ LBD16a), stimulated cell division when co-overexpressed with NF-YA1. ASL18, first characterized in the model plant Arabidopsis thaliana, is a transcription factor involved in establishing the asymmetry required for divisions of the pericycle founder cells that produce lateral root primordia (Goh et al., 2012). By phenotypic characterization of Lotus mutants, Soyano et al. (2019) conclude that ASL18 is involved in nodule development from very early stages. As expected, transcriptional and translational reporters indicate that ASL18 is expressed in early lateral root primordia derived from pericycle cells. Excitingly, expression was also found in nodule primordia localized to cortical cells. This result raises a key question: does ectopic expression of ASL18 contribute to the cortical cell divisions unique to legume nodule formation? To address this possibility, Soyano et al. (2019) examined DNA sequences within and around the ASL18 gene for NIN binding sites. An intronic NIN binding sequence is conserved in leguminous ASL18 but absent in non-leguminous orthologs. This finding suggests that evolution of a NIN binding site in ASL18 of an ancestral legume may have led to misexpression of ASL18 in cortical cells. Such an event would have caused a lateral root regulatory network, normally functioning in pericycle cells, to also function in cortical cells under control of
Developmental Cell
Previews cytokinin-responsive NIN, thereby leading to nodule formation. A recent report by Schiessl et al. (2019) also identified ASL18/LBD16 as a regulator of nodule formation in Medicago, a second species of legume. A comparative analysis indicated that the lateral root and nodule developmental pathways in this legume, despite differences in induction, have a large degree of overlap in formation and interpretation of local auxin accumulation. Shared genes include auxin-responsive transcriptional regulators and auxin biosynthesis genes such as STYLISH and members of the YUCCA and PLETHORA families. Thus, complementary studies in two legume species indicate that a co-opted lateral root developmental program functions ectopically in cortical cells and contributes to nodule organogenesis. These findings shed light on a years-old debate: is the legume nodule a modified lateral root or an organ sui generis (Hirsch et al., 1997)? This question was driven by observations that rhizobia induce lateral-root-like nodules derived from pericycle cells in a non-leguminous species. Now, the cortical derivation of legume nodules can be explained by evolution of a NIN binding site in ASL18 that is absent in non-leguminous species. It will be exciting to test if introduction of a NIN binding sequence to ASL18 in a non-legume will induce formation of a legume nodule.
The findings of Soyano et al. (2019) and Schiessl et al. (2019) indicate that nonleguminous plants likely harbor most of the genes required for nodulation. Further, Griesmann et al. (2018) report that the ability to form nodules was likely independently lost in many legume families rather than independently gained. Together, these results support the feasibility of engineering or re-engineering the ability to form nodules. The creation of staple crop species, such as cereals, with the ability to host nitrogen-fixing rhizobia will be key for reducing reliance on nitrate fertilizers, the large-scale application of which comes with great economic and environmental cost.
Griesmann, M., Chang, Y., Liu, X., Song, Y., Haberer, G., Crook, M.B., Billault-Penneteau, B., Lauressergues, D., Keller, J., Imanishi, L., et al. (2018). Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361. Published online May 24, 2018. https://doi. org/10.1126/science.aat1743. Hirsch, A.M., Larue, T.A., and Doyle, J. (1997). Is the legume nodule a modified root or stem or an organ sui generis? Crit. Rev. Plant Sci. 16, 361–392. Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44. Marsh, J.F., Rakocevic, A., Mitra, R.M., Brocard, L., Sun, J., Eschstruth, A., Long, S.R., Schultze, M., Ratet, P., and Oldroyd, G.E.D. (2007). Medicago truncatula NIN is essential for rhizobialindependent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase. Plant Physiol. 144, 324–335.
ACKNOWLEDGMENTS Work in the Benfey lab is funded by the Howard Hughes Medical Institute and the NIH (MIRA 1R35GM131725). DECLARATION OF INTERESTS P.N.B. is a founder of Hi Fidelity Genetics and a member of its scientific advisory board.
Schauser, L., Roussis, A., Stiller, J., and Stougaard, J. (1999). A plant regulator controlling development of symbiotic root nodules. Nature 402, 191–195. Schiessl, K., Lilley, J.L.S., Lee, T., Tamvakis, I., Kohlen, W., Bailey, P.C., Thomas, A., Luptak, J., Ramakrishnan, K., Carpenter, M.D., et al. (2019). NODULE INCEPTION recruits the lateral root developmental program for symbiotic nodule organogenesis in Medicago truncatula. Curr. Biol. 29, 3657–3668.e5.
REFERENCES Bensmihen, S. (2015). Hormonal control of lateral root and nodule development in legumes. Plants (Basel) 4, 523–547. Goh, T., Joi, S., Mimura, T., and Fukaki, H. (2012). The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins. Development 139, 883–893.
Soyano, T., Kouchi, H., Hirota, A., and Hayashi, M. (2013). Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus. PLoS Genet. 9, e1003352. Soyano, T., Shimoda, Y., Kawaguchi, M., and Hayashi, M. (2019). A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus. Science 366, 1021–1023.
Developmental Cell 52, January 6, 2020 7
Previews A Co-opted Regulator of Lateral Root Development Controls Nodule Organogenesis in Lotus Rachel Shahan1 and Philip N. Benfey1,* 1Duke University and Howard Hughes Medical Institute, Durham, NC 27701, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.12.009
Legumes, a subset of flowering plants, form root nodules in symbiosis with nitrogen-fixing bacteria. The regulatory network controlling nodule formation has remained mysterious. In a recent issue of Science, Soyano et al. (2019) demonstrate that co-option of an existing lateral root developmental program is used in Lotus for nodule organogenesis. Foundational questions in developmental biology concern the mechanisms underlying organ initiation and patterning. Postembryonic organogenesis is a feature unique to plants and is mediated by maintenance of stem cell populations in specialized niches. Two such niches, the primary shoot and root apical meristems, are formed in the developing embryo and are responsible for growth of the primary shoot and root, respectively. However, the post-embryonic formation of lateral organs is not directly governed by divisions of the primary meristems. Lateral roots are formed by divisions of specific, differentiated pericycle cells in response to environmental conditions (Malamy and Benfey, 1997). Intriguingly, the roots of several orders of flowering plants, most notably the legumes, are capable of forming an additional lateral organ, the nodule. Initiated by symbiotic interaction with nitrogen-fixing bacteria, legume nodules arise from divisions of differentiated cortical cells (Hirsch et al., 1997). Although lateral roots and nodules are both derived from primary root tissues, the evolutionary origin of the nodule and the molecular regulation of its organogenesis have long been mysterious. In a recent issue of Science, Soyano et al. (2019) show that a shared master regulator controls both lateral root and nodule organogenesis in Lotus japonicus, thereby providing evidence for co-option of an existing regulatory network for the development of a unique organ in legumes. Although all eudicots produce lateral roots, only a subset of genera within a single clade produce nodules and engage in symbiotic relationships with nitrogenfixing bacteria, collectively called rhizobia (Griesmann et al., 2018). Atmospheric ni-
trogen is abundant but unusable, while mineral forms are limiting for plant growth. Legumes such as pea, clover, and soy form nodules in order to host rhizobia in optimal conditions for nitrogen fixation. The host plant provides colonized rhizobia with a carbon source and, in exchange, benefits from the reduction of atmospheric nitrogen into ammonium. In this way, legumes are able to thrive in low-nitrogen terrestrial environments. Induction of nodulation in non-leguminous species could reduce nitrate fertilizer usage and revolutionize agriculture around the globe, but this goal first requires detailed knowledge of the molecular regulation of nodule organogenesis. Genetic approaches have identified several key regulators of nodule formation in legumes. One such nodulation-specific factor is Lotus japonicus NODULE INCEPTION (NIN), a transcription factor required for rhizobia infection and nodule organogenesis (Schauser et al., 1999; Marsh et al., 2007). NIN activates two genes that encode different subunits of a Nuclear Factor-Y (NF-Y)-binding protein complex. Ectopic expression of NIN and its two targets causes abnormal cell division during lateral root development and activates cortical cell division during nodule organogenesis, suggesting a link between nodule and lateral root developmental programs (Soyano et al., 2013). However, an enduring question remains: do nodule and lateral root organogenesis share a common regulatory pathway? Differences in response to cytokinin initially hinted that the answer is no. Cytokinin signaling is necessary and sufficient to induce nodule formation through induction of NIN but suppresses lateral root development (Bensmihen, 2015).
6 Developmental Cell 52, January 6, 2020 ª 2019 Elsevier Inc.
To dissect the molecular pathway underlying legume organogenesis, Soyano et al. (2019) examined existing ChIP-seq data to identify NIN target candidates with induced transcription in response to rhizobia. Only one candidate gene, ASYMMETRIC LEAVES 2-LIKE 18/ LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/ LBD16a), stimulated cell division when co-overexpressed with NF-YA1. ASL18, first characterized in the model plant Arabidopsis thaliana, is a transcription factor involved in establishing the asymmetry required for divisions of the pericycle founder cells that produce lateral root primordia (Goh et al., 2012). By phenotypic characterization of Lotus mutants, Soyano et al. (2019) conclude that ASL18 is involved in nodule development from very early stages. As expected, transcriptional and translational reporters indicate that ASL18 is expressed in early lateral root primordia derived from pericycle cells. Excitingly, expression was also found in nodule primordia localized to cortical cells. This result raises a key question: does ectopic expression of ASL18 contribute to the cortical cell divisions unique to legume nodule formation? To address this possibility, Soyano et al. (2019) examined DNA sequences within and around the ASL18 gene for NIN binding sites. An intronic NIN binding sequence is conserved in leguminous ASL18 but absent in non-leguminous orthologs. This finding suggests that evolution of a NIN binding site in ASL18 of an ancestral legume may have led to misexpression of ASL18 in cortical cells. Such an event would have caused a lateral root regulatory network, normally functioning in pericycle cells, to also function in cortical cells under control of
Developmental Cell
Previews cytokinin-responsive NIN, thereby leading to nodule formation. A recent report by Schiessl et al. (2019) also identified ASL18/LBD16 as a regulator of nodule formation in Medicago, a second species of legume. A comparative analysis indicated that the lateral root and nodule developmental pathways in this legume, despite differences in induction, have a large degree of overlap in formation and interpretation of local auxin accumulation. Shared genes include auxin-responsive transcriptional regulators and auxin biosynthesis genes such as STYLISH and members of the YUCCA and PLETHORA families. Thus, complementary studies in two legume species indicate that a co-opted lateral root developmental program functions ectopically in cortical cells and contributes to nodule organogenesis. These findings shed light on a years-old debate: is the legume nodule a modified lateral root or an organ sui generis (Hirsch et al., 1997)? This question was driven by observations that rhizobia induce lateral-root-like nodules derived from pericycle cells in a non-leguminous species. Now, the cortical derivation of legume nodules can be explained by evolution of a NIN binding site in ASL18 that is absent in non-leguminous species. It will be exciting to test if introduction of a NIN binding sequence to ASL18 in a non-legume will induce formation of a legume nodule.
The findings of Soyano et al. (2019) and Schiessl et al. (2019) indicate that nonleguminous plants likely harbor most of the genes required for nodulation. Further, Griesmann et al. (2018) report that the ability to form nodules was likely independently lost in many legume families rather than independently gained. Together, these results support the feasibility of engineering or re-engineering the ability to form nodules. The creation of staple crop species, such as cereals, with the ability to host nitrogen-fixing rhizobia will be key for reducing reliance on nitrate fertilizers, the large-scale application of which comes with great economic and environmental cost.
Griesmann, M., Chang, Y., Liu, X., Song, Y., Haberer, G., Crook, M.B., Billault-Penneteau, B., Lauressergues, D., Keller, J., Imanishi, L., et al. (2018). Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361. Published online May 24, 2018. https://doi. org/10.1126/science.aat1743. Hirsch, A.M., Larue, T.A., and Doyle, J. (1997). Is the legume nodule a modified root or stem or an organ sui generis? Crit. Rev. Plant Sci. 16, 361–392. Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44. Marsh, J.F., Rakocevic, A., Mitra, R.M., Brocard, L., Sun, J., Eschstruth, A., Long, S.R., Schultze, M., Ratet, P., and Oldroyd, G.E.D. (2007). Medicago truncatula NIN is essential for rhizobialindependent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase. Plant Physiol. 144, 324–335.
ACKNOWLEDGMENTS Work in the Benfey lab is funded by the Howard Hughes Medical Institute and the NIH (MIRA 1R35GM131725). DECLARATION OF INTERESTS P.N.B. is a founder of Hi Fidelity Genetics and a member of its scientific advisory board.
Schauser, L., Roussis, A., Stiller, J., and Stougaard, J. (1999). A plant regulator controlling development of symbiotic root nodules. Nature 402, 191–195. Schiessl, K., Lilley, J.L.S., Lee, T., Tamvakis, I., Kohlen, W., Bailey, P.C., Thomas, A., Luptak, J., Ramakrishnan, K., Carpenter, M.D., et al. (2019). NODULE INCEPTION recruits the lateral root developmental program for symbiotic nodule organogenesis in Medicago truncatula. Curr. Biol. 29, 3657–3668.e5.
REFERENCES Bensmihen, S. (2015). Hormonal control of lateral root and nodule development in legumes. Plants (Basel) 4, 523–547. Goh, T., Joi, S., Mimura, T., and Fukaki, H. (2012). The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins. Development 139, 883–893.
Soyano, T., Kouchi, H., Hirota, A., and Hayashi, M. (2013). Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus. PLoS Genet. 9, e1003352. Soyano, T., Shimoda, Y., Kawaguchi, M., and Hayashi, M. (2019). A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus. Science 366, 1021–1023.
Developmental Cell 52, January 6, 2020 7