NLPs - Crucial Regulators of Nitrate Signaling
Authors: Lekshmy Sathee, Jagadheesan B, Shailendra K Jha Nitrogen (N) is an essential macronutrient that impacts many aspects of plant physiology, growth, and development. Besides its nutritional role, N nutrient and metabolites act as signaling molecules that regulate the expression of a wide range of genes and biological processes, (Undurraga et al 2017). The critical role of nitrate as a signaling molecule has been established for several decades. However, the molecular mechanisms underlying the nitrate response have remained elusive, as the transcription factor that primarily responds to nitrate signals were not identified. The plant specific RWP-RK transcription factors are classified into two subfamilies: the NIN-like proteins (NLPs) and the RWP-RK domain proteins (RKDs). The NIN (for nodule inception) proteins were first identified as a regulator controlling development of symbiotic root nodules in the legume plant Lotus japonicus but were later found widely existing among plant species, including those that do not fix gaseous nitrogen (Chardin et al 2014).

The NLP family proteins differ greatly in size as observed from three inMedicago and Brachypodium, six in rice and nine inArabidopsis (Chardin et al. 2014). Functional studies in Arabidopsis using the genetic mutants and transgenic plants demonstrated that AtNLPs plays central role in orchestrating primary nitrate response (PNR) by binding to the nitrate-responsive cis-elements (Konishi and Yanagisawa 2013). Reports also suggest that the activity of NLPs is post translationally modulated by nitrate signaling. Recently, the nitrate-CPK (Ca2+-sensor protein kinase)-NLP network was identified, nitrate-coupled CPK signalling phosphorylates NLP transcription factors, specifically NLP7, and this signalling was crucial to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and cell proliferation (Liu et al 2017). AtNLP7 was shown to bind with key N pathway genes including ANR1, LBD37/38, NRT1.1, NRT 2.1, and NIA1, and thus able to moderate N assimilation and metabolism by either transcription activation or suppression of the downstream genes (Liu et al 2017).

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. The Arabidopsis NLP8 was found to be essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signaling, (Yan et al 2016)

Very little information is available on the protein protein interaction in regulating nitrate signaling. Guan et al (2017) report that teosinte branched1/cycloidea/proliferating cell factor1-20 (TCP20) and NLP6 and NLP7 binds to adjacent sites in the upstream promoter region of the nitrate reductase gene, NIA1, and physically interact under continuous nitrate and N-starvation conditions. Regions of these proteins necessary for these interactions were found to include the type I/II Phox and Bem1p (PB1) domains of NLP6&7, a protein-interaction module conserved in animals for nutrient signaling, and the histidine- and glutamine-rich domain of TCP20, which is conserved across plant species. Under N starvation, TCP20-NLP6&7 heterodimers accumulate in the nucleus, and this coincides with TCP20 and NLP6&7-dependent up-regulation of nitrate assimilation and signaling genes and down-regulation of the G2/M cell-cycle marker gene, CYCB1; 1. TCP20 and NLP6&7 also support root meristem growth under N starvation. These findings provide insights into how plants coordinate responses to nitrate availability, linking nitrate assimilation and signaling with cell cycle progression, (Guan et al 2017).

In conclusion, NLPS are master regulators of nitrate signalling and PNR in plants. They control wider range of genes related to nitrogen assimilation, nitrate transport, carbohydrate metabolism, hormone homeostasis and development. NLPs could be targets for altering nitrate signaling and improving NUE of crop plants.

References:

1. Chardin, C., Girin, T., Roudier, F., Meyer, C., Krapp, A., (2014) The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development. J Exp Bot 65(19): 5577–5587
2. Guan P, Ripoll, J.J., Wang, R., Vuong, L., Bailey-Steinitz, L.J., Ye, D. and Crawford, N.M., (2017) Interacting TCP and NLP transcription factors control plant responses to nitrate availability. PNAS, 114(9): 2419-2424
3. Konishi, M. and Yanagisawa, S., (2013) Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat Commun,; 4: 1617
4. Liu, K.H., Niu, Y., Konishi, M., Wu, Y., Du, H., Chung, H.S., Li, L., Boudsocq, M., McCormack, M., Maekawa, S. and Ishida, T., (2017) Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks. Nature, 545(7654): 311.
5. Undurraga, S.F., Ibarra-Henríquez, C., Fredes, I., Álvarez, J.M. and Gutiérrez, R.A., (2017). Nitrate signaling and early responses in Arabidopsis roots. J Exp Bot 68(10):2541-2551
6. Yan, D., Easwaran, V., Chau, V., Okamoto, M., Ierullo, M., Kimura, M., Endo, A., Yano, R., Pasha, A., Gong, Y. and Bi, Y.M., (2016) NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis. Nat Commun, 7:13179.




About Author / Additional Info:
Lekshmy S, Scientist ( Plant Physiology)at ICAR-IARI, New Delhi, Plant Physiologist working in the area of nitrogen use efficiency of crop plants.
Jagadheesan B, MSc Scholar, ICAR-IARI.
Shailendra K Jha, Scientist(Plant Breeding) at ICAR-IARI, New Delhi, Plant Breeder