Adjusting plant nutrient acquisition to fluctuating availability: transcriptional co-regulation of the nitrate and phosphate deprivation responses in roots

This article comments on: Safi A, Medici A, Szponarski W, Martin F, Clement-Vidal A, Marshall-Colon A, Ruffel S, Gaymard F, Rouached H, Leclercq J, Coruzzi G, Lacombe B, Krouk G. 2021. GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways. Journal of Experimental Botany 72, 3881–3901.

Native plant ecosystems are often nutritionally co-limited, especially in the two macroelements nitrogen (N) and phosphorus (P) (Fay et al., 2015). Substantial biomass increases are only expected when both elements are added, indicating synergisms between them. On evolutionary scales, nutritional deficiencies were the rule, rather than the exception, and modern heavily fertilized crops carry relicts of this in their genetics. Different plant species have different traits related to the acquisition of inorganic phosphate (P i , one of the least soil-mobile nutrients that is easily sorbed and fixed to clay particles) and nitrate (a highly mobile nutrient in soil), but common responses to low P and N exist, such as the anthocyanin production in leaves and the investment into roots, which results in a higher root/shoot ratio, mediated by phytohormone-driven redirection of photoassimilates. Nutritional interactions occur with other elements as well; for example, molybdenum and iron are linked with nitrate reduction, whereas P I mobilization mechanisms often co-solubilize iron, zinc, and manganese (Marschner, 2011). Function of NIGT1/HHOs in the P i deprivation response and the link with nitrate NIGT (NITRATE-INDUCIBLE GARP-TYPE TRANS-CRIPTIONAL REPRESSOR) genes, also known as HRS1 (HYPERSENSITIVITY TO LOW P i -ELICITED PRIMARY ROOT SHORTENING 1), and HHO (HRS1 HOMOLOG) genes coordinate nitrate and P i responses in Arabidopsis Maeda et al., 2018;Ueda et al., 2020b, c;Wang et al., 2020). Their names already indicate that they were identified in different nutritional contexts (Fig. 1).
NIGT1/HHO genes act as positive regulators of the P i starvation response. In brief, the master upstream regulators PHOSPHATE STARVATION RESPONSE 1-4 (PHR1-PHR4) bind to cis-elements in their promoters and activate them. In the presence of P i , PHRs form inactive complexes with SPX proteins (named after SYG1/Pho81/XPR1); the latter apparently sense the cellular P status. Under P i deficiency, PHRs are released and target cis-elements in promoters of P i uptake-, recycling-, and morphological adaptation-associated genes (Fig. 1). NIGT1/HHOs enhance transcription of P i uptake transporters (Wang et al., 2020) and repress SPX promoters, while PHRs moderately activate NIGT1/HHO genes (Maeda et al., 2018).
NIN-LIKE PROTEIN 6/7 (NLP6/7) transcription factors, master regulators of the nitrate response, promote nitrate uptake/assimilation genes and NIGT1 expression (Fig. 1). This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) | 3501 NIGT1/HHOs dimerize with each other and with more distant HHO proteins; this determines specificity and affinity for targets (Ueda et al., 2020b).
In a feedback loop, NIGT1/HHO proteins repress nitrate uptake upon P i deprivation, and vice versa. P i uptake is repressed in the absence of nitrate, as SPX promoters are then not repressed by NIGT1/HHOs (Ueda et al., 2020c). Safi et al. showed that double and quadruple nigt1/hho mutants had elevated high affinity nitrate uptake concomitant with the de-repression of high affinity nitrate transporters. This correlates with an improved growth under controlled conditions with sufficient P i (Safi et al., 2021). Wang et al. (2020) also previously reported an improvement of low affinity uptake in hho mutants, probably due to NRT1.1. One should, however, keep in mind that Col-0, the genotype used in these studies, is quite inefficient using nitrogen compared with other Arabidopsis accessions (Chardon et al., 2010;Menz et al., 2018). Even though there is functional diversification within the NIGT1/HHO family among monocots and dicots (Ueda et al., 2020a), the transcriptional co-repression of nitrate influx by P i deficiency seems to be conserved in maize (Wang et al., 2020), and P i starvation control by nitrate is conserved in wheat and rice (Medici et al., 2019). Still, extrapolation to field-grown crops should be done with caution, as biomass increases under control conditions rarely translate into the field. The quadruple nigt1/hho mutant was impaired in P i uptake under P deficiency, but not when sufficient P was available (Ueda et al., 2020c). Benefits in nitrate nutrition are thus compromised by negative effects on P acquisition, and crops that fail to adjust to low P i are undesirable, even with improved nitrate acquisition. It is possible that complex feedback-regulated networks also help plants to explore temporal variations in nutritional availability (e.g. in field environments). As long as the underlying element is not rate limiting for growth, Arabidopsis profits from temporal reduction of external P i , compared with static supply (Fig. 2).

Connecting nutrient deprivation/starvation responses with ROS
Potassium, N, or P i deprivation are known to increase H 2 O 2 in distinct areas of Arabidopsis roots; this and the misregulation of starvation-responsive nitrate and phosphate transporter genes in an NADPH oxidase mutant (atrbohC) provided some initial hints of ROS involvement in nutrient-specific responses (Shin et al., 2005). Enforcement of the transcriptional nitrate starvation response by glutaredoxins (Jung et al., 2018;Ehrary et al., 2020) and inhibition by chemical ROS scavenging point to an involvement of ROS homeostasis under fluctuating nutrient conditions. This is supported by Safi et al. (2021), who conclude that 86% of the direct (repressed) targets of HRS1 in protoplasts were dependent on the nitrate context (then HRS1 acted mainly as a repressor of heat shock genes). In complex targeted gene categories, the redox metabolism was over-represented. H 2 O 2 production was not compromised in a quadruple nigt1/ hho mutant, but HRS1/HHO1 overexpression suppressed H 2 O 2 evolution upon nitrate deprivation. ROS signalling must not reach toxic levels within cells (Miller et al., 2018) and may participate in the initiation of the response, rather than act as an unwanted by-product of long-term N starvation due to insufficient production of proteins involved in scavenging. Furthermore, NIGT1/HHO genes are highly specific to nitrate, rather than ammonium. Rapid responses within hours to nitrate deprivation are completely absent when ammonium is removed from the nutrient solution, and transcriptional changes are seen before nitrate levels drop in the tissue (Menz et al., 2016). We therefore associate the NIGT1/HHO repressors and ROS with nitrate deprivation, rather than with N starvation responses (which occur later). Interestingly, the mRNA of HRS1 declined quickly without nitrate, but was stabilized by NO 3 -, pointing to post-transcriptional control (Menz et al., 2016). It may be interesting to ask if there is a link to ROS by carrying out in silico analysis using other datasets from previous transcriptomic studies, for example on AtNIGT1.2/AtHHO2 overexpressors Wang et al., 2020).

Nutritional co-regulation is highly complex
Further crosstalk between N and P might depend on LATERAL ORGAN BOUNDARY DOMAIN transcription factors (LBD37-LBD39), another group of redundant repressors of the N deprivation response that are highly up-regulated by diverse N sources, preferentially nitrate. Their main function is to repress anthocyanin biosynthesis in nitrate-sufficient conditions. Their transcripts are repressed in -N, leading to anthocyanin coloration of shoots (and ROS scavenging) in severely -N-stressed plants (Rubin et al., 2009). LBD transcripts may be repressed in -P (Rubin et al., 2009) and less anthocyanin accumulated in the nigt1/hho quadruple mutant with sufficient N (Ueda et al., 2020c), suggesting crosstalk with LBDs in -P (Fig. 1).
In summary, NIGT1/HHOs are central hubs in the crosstalk between nitrate and phosphate deprivation responses. How NIGT1/HHOs co-regulate these nutritional responses  (Menz et al., 2018) with 1 mM ammonium nitrate and P i for 52 d (control), or 32 d and then transiently transferred to 50 µM N (low N) or 50 µM P i (low P) for 2 weeks. These plants were then resupplied with full nutrient solution with 1 mM P i and N for an additional week (all other nutrients were kept at the same level). (B) Shoot biomass, (C) root biomass, (D) P in rosette, (E) N in rosette, (F) root/ shoot ratio. Note that the growth was transiently impaired in low N, but not in low P (as P i was still sufficient for maximal growth). After the transient treatment, 'low P' plants grew much better than controls that achieved high P throughout. This is an example showing that nutrient fluctuations, if not growth limiting, can have beneficial effects on plant growth (and possibly nitrate acquisition). It is possible, but not known, whether NIGT1/HRS signalling is responsible for this effect. We note that experiments under laboratory conditions with agar plate/hydroponic systems often involve P i concentrations >100-fold higher than typical soil P i solution concentrations (<10 µM P i ).
in crop roots and whether these can be targeted to generate more nutrient use-efficient crops is an interesting task for the future. Overexpression of various nitrate transporters increased nitrate acquisition and yield apparently independently of P, not only in laboratory studies, but even in the field (e.g. Wang et al., 2018), supporting the added value of studying these genes and proteins at a fundamental level.