Nitrate Induction of Primary Root Growth Requires Cytokinin Signaling in Arabidopsis thaliana Pamela A. Naulin, Grace I. Armijo, Andrea S. Vega, Karem P. Tamayo, Diana E. Gras, Javiera de la Cruz and Rodrigo A. Guti  errez * Departamento de Gen etica Molecular y Microbiología, FONDAP Center for Genome Regulation, Millennium Institute for Integrative Biology (iBio), Pontificia Universidad Cat olica de Chile, Santiago 8331150, Chile *Corresponding author: E-mail, rgutierrez@bio.puc.cl; Fax, (56-2)-2-354-2185. (Received February 25, 2019; Accepted October 16, 2019) Nitrate can act as a potent signal to control growth and development in plants. In this study, we show that nitrate is able to stimulate primary root growth via increased meri- stem activity and cytokinin signaling. Cytokinin perception and biosynthesis mutants displayed shorter roots as com- pared with wild-type plants when grown with nitrate as the only nitrogen source. Histological analysis of the root tip revealed decreased cell division and elongation in the cyto- kinin receptor double mutant ahk2/ahk4 as compared with wild-type plants under a sufficient nitrate regime. Interestingly, a nitrate-dependent root growth arrest was observed between days 5 and 6 after sowing. Wild-type plants were able to recover from this growth arrest, while cytokinin signaling or biosynthesis mutants were not. Transcriptome analysis revealed significant changes in gene expression after, but not before, this transition in con- trasting genotypes and nitrate regimes. We identified genes involved in both cell division and elongation as potentially important for primary root growth in response to nitrate. Our results provide evidence linking nitrate and cytokinin signaling for the control of primary root growth in Arabidopsis thaliana. Keywords: Arabidopsis thaliana • Cytokinin • Nitrate • Primary root growth. Introduction Nitrogen (N) is an essential macronutrient required for plant growth and development. N availability is a critical factor for agricultural productivity (Frink et al. 1999). High-yield crop pro- duction is dependent on the application of large quantities of nitrogenous fertilizers over time. However, a large fraction of N is not directly absorbed by plants and is lost by leaching (Hirel et al. 2011), leading to detrimental effects on the ecosystem and human health. Understanding how plants sense and respond to N is critical for developing strategies to improve its use for sustainable agriculture. Considerable progress has been made in recent years to dissect the nitrate signaling pathway in plants. The dual- affinity nitrate transporter NRT1.1/NPF6.3 has been proposed as a nitrate sensor (Noguero and Lacombe 2016), essential for multiple independent signaling pathways in response to nitrate in Arabidopsis thaliana (Bouguyon et al. 2015). In addition, ni- trate treatment induces a rapid increase in cytoplasmic and nuclear Ca 2þ levels (Riveras et al. 2015, Liu et al. 2017), indicat- ing that Ca 2þ is an important second messenger in the nitrate signaling pathway downstream of NRT1.1/NPF6.3. One of the direct consequences of Ca 2þ increase is the change in protein phosphorylation status, connecting the influx of calcium with kinases that active transcription factor targets to command the primary nitrate response (Liu et al. 2017). To survive with heterogeneous N availability in the soil, plants evolved elaborate developmental strategies to optimize its acquisition (Vidal and Gutierrez 2008, Bouguyon et al. 2012, Nacry et al. 2013, Kiba and Krapp 2016, Fredes et al. 2019). In A. thaliana plants, nitrate modulates primary and lateral root growth (Walch-Liu et al. 2005, Walch-Liu et al. 2006a, Gifford et al. 2008, Krouk et al. 2010, Vidal et al. 2010, Bouguyon et al. 2012, Forde 2014, Krapp et al. 2014, Ruffel et al. 2014, Kiba and Krapp 2016). Depending on experimental conditions, nitrate can have both positive and negative effects on primary root growth (Crawford and Glass 1998, Bouguyon et al. 2012, Wang et al. 2012, Krapp et al. 2014, Ruffel et al. 2014, O'Brien et al. 2016). Root length and lateral root formation are modulated by the cross talk of auxin, cytokinin and ethylene, which are the keys for N signaling. At the same time, N can control hormone biosynthesis, signaling and transport in plants. For instance, NRT1.1/NPF6.3 is involved in the repression of lateral root growth at low nitrate availability by the modification of auxin transport (Krouk et al. 2010). Furthermore, lateral root initi- ation and emergence are controlled by microRNA167/auxin response factor 8 (ARF8) regulatory motif (Gifford et al. 2008) and a regulatory module that includes miR393 and the auxin receptor AFB3 was shown to mediate both lateral and primary root growth in response to nitrate treatments in A. thaliana (Vidal et al. 2010). On the other hand, there is evidence that ethylene may participate in the nitrate regulation of lateral root growth by modulating transporters in response to high nitrate concentration (Tian et al. 2009). Abscisic acid (ABA) was also related with lateral root inhibition by high nitrate since this effect was significantly reduced in three ABA insensitive Plant Cell Physiol. 61(2): 342–352 (2020) doi:10.1093/pcp/pcz199, Advance Access publication on 15 November 2019, available online at https://academic.oup.com/pcp # The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. 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