Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

Plant and Cell Physiology Advance Access published January 28, 2016

Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1

Subject areas: environmental and stress responses; membrane and transport

Corresponding author:

Christophe Maurel Biochimie et Physiologie Moléculaire des Plantes, Bât. 7, Campus INRA/SupAgro, 2 place Viala F-34060 Montpellier Cedex 2 France Tel : + 33 499 612011 Fax : + 33 467 525737 E-mail : [email protected]

Figure number:

Black and white :

5 (Figs. 1, 3, 5, 6 and 7)

Colour :

2 (Figs. 2 and 4)

Supplementary materials: 5 figures. 1 table

© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]

1

Downloaded from http://pcp.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 3, 2016

Running head: Nitrate regulates root hydraulic conductivity

Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1

Authors: Guowei Li1,2, Pascal Tillard1, Alain Gojon1 and Christophe Maurel1*

Addresses : 1

Biochimie et Physiologie Moléculaire des Plantes, Unité Mixte de Recherche 5004,

INRA/CNRS/Montpellier SupAgro/Université Montpellier, F-34060 Montpellier, Cedex 2, France.

2

Bio-Tech Research Center, Shandong Academy of Agricultural Sciences; Shandong

Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100, PR China

Abbreviations: HATS, high-affinity transport system; LATS, low-affinity transport system; LN, low nitrate; Lpr, root hydraulic conductivity; N, nitrogen; NH4+, ammonium; NO3-; nitrate; PIP, plasma membrane intrinsic protein; q-PCR, quantitative real-time RT-PCR; ST, standard.

2


Running title: Nitrate regulates root hydraulic conductivity

Abstract The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3-), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To

NO3- transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1, nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3- conditions. First, a strong positive relationship between Lpr and NO3- accumulation, in shoots rather than in roots, was revealed. Second, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role of high-affinity NO3- transporter NRT2.1 in increasing Lpr. These results indicate that NO3- signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative RT-PCR and ELISAs were used to investigate the effects of NO3- availability on plasma membrane aquaporin (PIP) expression. Whereas PIP regulation mostly occurs at post-translational level in wild-type plants, a regulation of PIPs at both transcriptional and translational levels was unmasked in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3- involves novel shoot-to-root signaling and NRT2.1-dependent functions.

Keywords: aquaporin, Arabidopsis thaliana, nutrient, root, shoot-to-root signaling, water transport.

3


explore the effects and mode of action of NO3- on Lpr, we used an extended set of

Introduction Higher plants are autotrophic organisms that synthesize organic components using inorganic molecules absorbed from their environment. Whereas atmospheric carbon dioxide is used as a major source for carbon fixation, nitrogen (N) assimilation,

(NO3-) and ammonium (NH4+) from the soil. NO3-, which represents the main N source for many plants species, is taken up by root cells using low-affinity or high-affinity transport systems (LATS and HATS, respectively), depending on its external concentration. In Arabidopsis thaliana, the molecular components of the LATS identified to date are NRT1.1 (NPF6.3) and NRT1.2 (NPF4.6), which are members of the NRT1/PTR (NPF) family of transporters (Léran et al. 2014; Wang et al. 2012). In contrast, the HATS mostly correspond to transporters of the NRT2 family (NRT2.1, 2.2, 2.4 and 2.5) (Krapp et al. 2014; Lezhneva et al. 2014), among which NRT2.1 plays a predominant role as it can account for up to 75% of the total HATS activity (Filleur et al. 2001; Gojon et al. 2009; Remans et al. 2006b). Following uptake from the external medium, NO3- can either be stored into vacuoles, reduced to nitrite in root cells by cytosolic NIA1 and NIA2 nitrate reductases, or loaded into the xylem, in particular by the NRT1.5 (NPF7.3) efflux carrier (Lin et al. 2008), for translocation to the shoot. More than a simple mineral nutrient, NO3- plays a central signaling role, to regulate processes related to its own transport, storage and assimilation and to coordinate them with a wide range of processes, including carbon metabolism, secondary metabolism, hormone signaling, germination and root development (Bouguyon et al. 4


another crucial pathway of plant metabolism, mostly relies on absorption of nitrate

2012; Gojon et al. 2011; Krapp et al. 2014; Krouk et al. 2011; Stitt 1999). Although most of molecular mechanisms of NO3- signaling are unknown, a role for NIN-LIKE PROTEIN (NLP) transcription factors has recently been identified (Marchive et al. 2013). It was also proposed that NO3- sensing involves NRT1.1, acting as a

2010; Krouk et al. 2006). A signaling role has also been proposed for NRT2.1 in the NO3- regulation of root development (Remans et al. 2006b; Zheng et al. 2013). Plant water transport, which promotes convection flow of soil nutrients to the root surface, and their uptake and allocation throughout the plant body, is intimately linked to plant mineral nutrition in general, and to N assimilation in particular. The water transport capacity of the root system (root hydraulic conductivity; Lpr) is determined in large part by the function of aquaporin water channels (Peret et al. 2012; Sutka et al. 2011; Tournaire-Roux et al. 2003). Aquaporins with more than 30 members in higher plants fall into 4 subgroups (PIPs, TIPs, NIPs, SIPs) in Arabidopsis, maize and rice (Chaumont et al. 2001; Johanson et al. 2001; Sakurai et al. 2005). Up to 3 additional subgroups (XIPs, GIPs and HIPs) with as yet unknown functions, were recently found in moss, protozoa, fungi and certain higher plant species (Anderberg et al. 2011; Anderberg et al. 2012; Lopez et al. 2012). Converging physiological and genetic evidences have established the contribution of PIPs to Lpr (Peret et al. 2012; Postaire et al. 2010; Tournaire-Roux et al. 2003). The latter is subjected to regulation by numerous hormonal and environmental stimuli including abscisic acid and auxin, water or nutrient availability or oxidative stresses. These regulations are mediated 5


transceptor, with a dual transport/signaling function (Gojon et al. 2011; Krouk et al.

through multiple mechanisms involving transcriptional aquaporin gene regulation, posttranslational control of aquaporin sub-cellular trafficking and opening and closing (gating), and regulated protein degradation (Li et al. 2014; Maurel et al. 2015). A profound interplay between N availability (mainly NO3-) and water absorption has

species investigated, such as Arabidopsis, sunflower, maize, wheat, lotus and rice, limited NO3- availability resulted in a dramatic (>50%) reduction in Lpr (Carvajal et al. 1996; Clarkson et al. 2000; Di Pietro et al. 2013; Gloser et al. 2007; Ishikawa-Sakurai et al. 2014; Prosser et al. 2006; Radin and Boyer 1982).

Depending on species,

these changes which are reversible upon NO3- re-supply, can occur over a couple of hours (Lotus japonicus, sunflower) or days (Arabidopsis)(Clarkson et al. 2000; Di Pietro et al. 2013; Gloser et al. 2007). Despite its potentially high agronomic importance, the mode of action of NO3- on root water transport is largely unknown. Firstly, the nature of the primary signal is still debated. Early experiments relying on inhibitors of key enzymes of the N assimilation pathway suggested that NO3assimilation products rather than nitrate itself may trigger root signaling leading to enhanced hydraulics (Barthes et al. 1996; Gloser et al. 2007). Yet, the specificity of sodium tungstate used as a nitrate reductase inhibitor has recently been questioned in cucumber and tomato, due to unexpected inhibitory effects on tissue NO3accumulation (Gorska et al. 2008a). The additional finding that intracellular injection of NO3- enhances cell hydraulic conductivity over a couple of min and can overcome its inhibition by sodium tungstate rather supports the idea that intracellular NO3- acts as a 6


been reported in numerous plant species (Cramer et al. 2009). In virtually all plant

primary signal (Gorska et al. 2008a). The molecular and cellular mechanisms by which NO3- acts on Lpr also remain unclear. Nitrate was shown to alter aquaporin expression in tomato (Wang et al. 2001) and recent studies in rice indicate that N (NH4NO3) alters Lpr through changes in aquaporin abundance (Ishikawa-Sakurai et al.

was observed in maize (Gorska et al. 2008b). In Arabidopsis, a phosphoproteomic analysis suggested a general role of aquaporin phosphorylation in stimulus-induced regulation of Lpr (Di Pietro et al. 2013). It was also proposed that NO3- deprivation results in a drop in cytosolic pH, which in turn would contribute to H+-dependent PIP gating (Gloser et al. 2007). In this work, we took a reverse genetics approach to explore the impact and mode of action of NO3- on water transport. To identify the key steps of NO3- utilization by the plant which affect Lpr, we used an extended set of NO3- transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1, nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3- conditions. Our data revealed a strong relationship between Lpr and plant NO3- accumulation together with a specific role of high-affinity NO3- transporter NRT2.1 in determining Lpr. The modes of Lpr regulation by NO3availability and NRT2.1 were investigated by monitoring PIP expression at both transcriptional and translational levels.

7


2014). Yet, no correlation between NO3--dependent Lpr and aquaporin expression

Results NO3-and water uptake activity in roots of NO3-transport and assimilation mutants Root NO3- uptake activity of nrt1.1-1, nrt1.2-1, nrt1.5-1, nrt2.1-1 and nia plants grown

short-term

15

NO3- influx. Total and high affinity (HATS) NO3-uptake capacities were

determined from influx assays at 5 mM and 0.5 mM

15

NO3-, respectively. Low affinity

transport capacity (LATS) was deduced from the difference of total and HATS transports. In nrt1.1, LATS activity was decreased with respect to wild-type (Col-0) whereas HATS was increased (Supplemental Fig. S1A), in agreement with previous observations (Muños et al. 2004). In contrast, no alteration in NO3- uptake capacity was observed in nrt1.2 compared to wild-type (WS) (Supplemental Fig. S1B). The nrt1.5 mutant showed a marginal decrease in total root NO3- influx, which could be accounted for by a specific defect in HATS (Supplemental Fig. S1C). In contrast, nrt2.1-1 plants displayed a dramatic reduction in root NO3- uptake, due to both lowered HATS and LATS activities (Supplemental Fig. S1C). Finally, no significant difference in NO3- uptake activity was found between nia mutant and wild-type plants (Supplemental Fig. S1D). With the exception of nrt1.2 plants (Huang et al. 1999), these data are strongly consistent with those previously reported for the nrt1.1, nrt2.1 and nia genotypes (Filleur et al. 2001; Muños et al. 2004; Unkles et al. 2004) , and mostly highlight the central role of NRT2.1 in root NO3- acquisition in Arabidopsis. The pressure chamber technique was then used to evaluate the root water 8


in standard (ST) N conditions (5 mM external NO3-) was investigated by measuring

transport capacity (Lpr) of the same set of genotypes. Fig. 1 shows that the Lpr of nrt1.1-1, nrt1.2-1, nrt1.5-1 and nia grown under ST conditions was not changed by reference to their respective wild-type controls. In contrast, nrt2.1-1 showed a nearly 30% reduction in Lpr by comparison to Col-0 (Fig. 1).

concentrations Because the nrt2.1-1 mutant was by far the genotype most affected on root NO3uptake, it is possible that the specific Lpr phenotype of this mutant could be the consequence of a reduced NO3- accumulation in the plant. We therefore investigated to which extent Lpr can be linked to external NO3- availability or to the amount of NO3accumulated in roots and/or shoots. For this, Col-0, nrt1.1-1, nrt1.5-1, nrt2.1-1 and nia plants were grown for 18 d under ST conditions, prior to transfer for 3 d in a hydroponic solution containing various NO3- concentrations (0, 0.5, 5 or 10 mM). In Col-0, a large variation of NO3- content in shoots and roots was observed, in relation to external NO3- availability (Fig. 2). From 0 to 10 mM NO3- treatments, the range of NO3- accumulation in shoots varied from 0 to 1.2 mmol/gDW and was somewhat lower in roots (0 to 0.8 mmol/gDW). The corresponding changes in NO3- accumulation recorded in the various mutants fit with those expected from their NO3- transport and assimilation phenotypes (Supplemental Fig. S1). Both nrt1.1-1 and nrt1.5-1 mutants, which display little alteration of root NO3- uptake as compared to Col-0 showed NO3accumulation profiles similar to that of Col-0 (Fig. 2). In contrast, the nia plants deficient for nitrate reductase showed, with respect to Col-0, a significant 9


Lpr and NO3- content in shoots and roots of plants grown under various NO3-

over-accumulation of NO3- in shoots, especially at low external NO3- (0.5 mM). The nrt2.1-1 plants showed an opposite phenotype with NO3- concentrations in both shoots and roots at low NO3- availability much lower than those measured in Col-0. In all genotypes, there was virtually no NO3- accumulation in both shoots and roots after

Dose-dependent effects of external NO3- on Lpr were also examined. In all genotypes, a dual pattern was observed, with increasing Lpr from 0 mM to 0.5 or 5 mM, and a relative inhibition of Lpr at higher NO3- concentration. Thus, depending on genotypes, Lpr showed a maximal value at either 0.5 mM (nrt1.5-1, nia) or 5 mM external NO3- (Col-0, nrt1.1-1, nrt2.1-1) (Fig. 2). In all 5 genotypes investigated, however, the lowest values were always observed in NO3- free conditions. These data indicate that the response of root water transport to changes in external NO3concentration was not drastically affected by the mutations investigated. They also establish that the nrt2.1-1 mutant had Lpr values significantly lower than in Col-0, at all external NO3- conditions investigated. This phenotype was confirmed for both low nitrate (LN; 0.5 mM NO3-) and ST conditions in a second allelic mutant, nrt2.1-2 (Fig. 3), which also shows severe defects in both HATS and LATS NO3- uptake activity (Supplemental Fig. S2). Correlation between Lpr and nitrate content in shoots and roots The large variations of Lpr and NO3- content in shoots and roots seen across the various NO3- treatments and genotypes (Fig. 2) were used to investigate a possible relationship between Lpr and tissue NO3- content. The supra-optimal effects of high 10


growth for 3 d in a NO3--free solution (Fig. 2).

NO3- (10 mM) on Lpr may be due to an inhibitory process that is mechanistically different from the stimulation observed for external NO3- concentrations up to 5 mM. In support for this, stronger correlation between Lpr and shoot or root NO3- content was observed when the data from 10 mM NO3- treatments were not considered. In this

content (R2 = 0.5676; p = 0.0012) (Fig. 4A). A significant correlation was also observed between Lpr and root NO3- content (R2 = 0.3563; p = 0.0188) (Fig. 4B). Multiple linear regression analysis yielded a higher coefficient of determination (R2 = 0.6734) but, in this case, only shoot NO3- content was significantly correlated to Lpr (p = 0.0051). In addition, Spearman's rank correlation was significant in the case of shoot NO3- content (p= 0.006), but not of root NO3- content (p = 0.07). These results suggest that if the changes in Lpr reported in Figs. 1 and 2 reflect a causal relationship between tissue NO3- content and Lpr then shoot NO3- content, rather than root NO3content, exerts a predominant role. However, shoot NO3- content was not the only factor involved in determining changes in Lpr in our experiments. Indeed, Lpr values in nrt2.1-1 plants were always lower than in the other four genotypes, regardless of shoot NO3- content (Fig. 4A) and the effects of NRT2.1 on root water transport were seen even when NO3- was not present in the medium. Reduced Lpr in nrt2.1-1 plants also could not be explained by differences in growth as compared to other genotypes (see Supplemental Table S1). Although root dry weight (DWr), used as a proxy of root size for Lpr calibration, was significantly smaller in nrt2.1-1 than in Col-0, our data set did not show any correlation between Lpr and DWr (Supplemental Fig. S3) nrt2.1 also 11


case, the most significant linear correlation was observed between Lpr and shoot NO3-

had a lower shoot dry weight (DWs) than Col-0. However, Lpr of nrt2.1 was lower than in nrt1.1, another genotype with similarly low DWs, indicating that reduced Lpr was not simply due to a reduced water demand from the shoots (Supplemental Fig. S4). Thus, nrt2.1-1 plants appear to display a true alteration in root water transport capacity that

changes in growth. PIP gene expression in roots of Col-0 and nrt2.1 plants grown under contrasting external nitrate conditions. To further explore the modes of Lpr regulation by NO3- and NRT2.1, we used quantitative real-time RT-PCR (q-PCR) and investigated the transcript abundance of all 13 PIP genes in roots of WT and nrt2.1 plants grown under ST and LN conditions. External NO3- had no major effects on PIP transcript abundance in Col-0 (Fig. 5 and Supplemental Fig.S5). However, lower transcript abundance was identified for several PIP genes including PIP1;1, PIP1;2, PIP2;1, PIP2;3 and PIP2;7 in the two nrt2.1 alleles as compared to Col-0 under ST conditions (Fig. 5 and Supplemental Fig. S5). This reduced expression could account for the lower Lpr of nrt2.1 plants in these conditions (Figs. 1 and 2). Strikingly, expression of PIP1;1, PIP1;2, PIP2;1, PIP2;2 and PIP2;3 in nrt2.1 plants was further markedly reduced under LN as compared to ST conditions, while such effects were not observed in Col-0 (Fig. 5). No significant variation was observed in other PIPs (Supplemental Fig. S5). To further delineate the modes of Lpr regulation in nrt2.1 mutants, we analyzed the relationship between Lpr and PIP transcript abundance under ST and LN 12


is not a consequence of decreased NO3- uptake and accumulation, or of associated

conditions, adding as a reference PIP transcript abundance in Col-0 under ST conditions. Four PIP genes, including PIP1;1, PIP1;2, PIP2;1 and PIP2;3, showed a strong positive correlation between transcript abundance and Lpr (p values < 0.05) (Fig. 6A). Thus, the reduced Lpr of nrt2.1 under ST conditions, and its further inhibition

(PIP1;1, PIP1;2, PIP2;1, PIP2;3). By contrast, the transcript abundance of PIP1;4 and PIP2;6 showed a tendency to negative correlation with Lpr (Fig. 6B). PIP protein abundance in roots of Col-0 and nrt2.1 plants grown under contrasting external nitrate conditions. To get further insights into PIP regulation by NO3- and NRT2.1, we investigated the protein abundance of PIPs in response to external NO3-, in both Col-0 and nrt2.1 plants, and performed ELISAs with antibodies directed against PIP1s (PIP1;1-PIP1;4) and PIP2s (PIP2;1-PIP2;3). Consistent with the lack of effects on transcript abundance, external NO3- did not alter PIP1 and PIP2 abundance in Col-0 (Fig. 7A and B). The abundance of PIP1s in the two nrt2.1 mutant alleles was significantly lower than that in Col-0, in both ST and LN conditions (Fig. 7A). In contrast, the abundance of PIP2s in nrt2.1 mutants was reduced with respect to that in Col-0, only in LN conditions (Fig. 7B). Overall, low external NO3- induced lower PIP1 and PIP2 abundance in the two nrt2.1 mutant alleles, in agreement with effects seen on transcript abundance. In addition, the whole set of data indicated a positive correlation between Lpr and PIP1 and PIP2 abundance (Fig. 7C and D).

13


at LN may both be mediated through gene regulation of a subset of PIP genes

Discussion Physiological studies have shown that whereas ammonium has an essentially negative impact (Guo et al. 2007), NO3- exerts beneficial effects on root water uptake in numerous plant species. In the present work, we took a reverse genetic approach

and signaling. The various genetic backgrounds available in Arabidopsis were used to analyze root hydraulic regulation in a broad range of external and internal nitrate configurations. Our data highlight three main points. First, they indicate a strong relationship between Lpr and NO3- accumulation in shoots. Second, they unravel an original and nitrate-independent role of NRT2.1 in determining Lpr. Finally, they reveal how NO3- and NRT2.1 alter Lpr by regulation of PIP expression at both transcriptional and translational levels. What is the genuine signal acting on Lpr? Nitrate affects many physiological and developmental processes in plants, including in addition to water transport, its own assimilation pathway, carbon and secondary metabolisms, germination, and root and shoot development. These responses can be triggered by NO3- acting as a signal per se (Bouguyon et al. 2012; Krapp et al. 2014; Krouk et al. 2011; Stitt 1999), or through signal molecules related to downstream products of NO3- assimilation and representative of the organic N status of the whole plant (Forde 2002; Gojon et al. 2009; Nacry et al. 2013). For most plant responses to NO3-, specific N signaling pathways and associated molecular transducers have been identified (Bouguyon et al. 2012; Krapp et al. 2014; Krouk et al. 2011; Stitt 1999). 14


to further dissect the hydraulic effects of NO3- and target its -transport, assimilation

Although a few studies have addressed the modes of root water transport regulation by NO3-, it is still disputed whether NO3- acts directly through specific signaling mechanisms, or indirectly through metabolic derivatives. For instance, the use of tungstate as a nitrate reductase inhibitor led to the conclusion that NO3- exerts direct

products in maize (Barthes et al. 1996). However, tungstate can have confounding side effects: it prevented plant NO3- accumulation in maize and cucumber (Gorska et al. 2008a; Gorska et al. 2008b) and reduced aquaporin expression in maize (Gorska et al. 2008b). Our data provide two lines of evidence that NO3- signaling is involved in regulating Arabidopsis Lpr. First, both the absolute values of Lpr and its response to changes in NO3- availability were unaffected by nitrate reductase deficiency in the nia mutant, suggesting that NO3- itself, rather than its assimilation products, is the main signal driving Lpr. Second, the most dramatic changes in Lpr were recorded in the nrt2.1 mutants. Most interestingly, Lpr defects in nrt2.1 plants were also observed in the absence of NO3-, indicating that effects of NRT2.1 on root hydraulics can be uncoupled from its NO3- transport function. Similar characteristics were taken as an evidence for a direct signaling role of NRT2.1 during lateral root development in Arabidopsis (Little et al. 2005; Remans et al. 2006b). Since NRT2.1 gene expression is enhanced at intermediate NO3- concentration (0.1-0.5 mM) while being repressed at high NO3- (5-10 mM) (Zhuo et al. 1999), our data also indicate that NRT2.1 effects on Lpr cannot be directly linked to its gene expression level. NRT1.1 is now considered 15


effects on Lpr in tomato (Gorska et al. 2008a) whereas it acts through its assimilation

as a main NO3- sensor triggering many of the above mentioned responses to NO3(Bouguyon et al. 2015; Gojon et al. 2011; Krapp et al. 2014; Wang et al. 2012). Surprisingly, nrt1.1 plants occasionally showed a reduced Lpr (see Fig. 2) but had no reproducible water transport phenotype (Fig. 1), suggesting that NRT1.1 plays a little

Another unexpected result, as illustrated by the correlation analyses in Fig. 4, was that the dominating signal in regulating Lpr would not be external nor root-accumulated NO3-, but rather the shoot NO3- pool. This hypothesis is consistent with the conclusions of split-root experiments in rice (Ishikawa-Sakurai et al. 2014) showing that regulation of root aquaporin gene expression by NO3- was systemic and not local for most (5 out of 7) of PIP or TIP NO3-- responsive genes. Shoot-borne signals acting on root hydraulics have also been hypothesized in the context of Lpr regulation by transpiration or wounding (Ishikawa-Sakurai et al. 2014; Laur and Hacke 2013; Liu et al. 2014; Vandeleur et al. 2014). In addition, shoot-to-root NO3- signaling has been evidenced for regulation of root development in Arabidopsis (Forde 2002; Zhang et al. 1999), but the underlying mechanisms are unknown. The NRT1.1 transceptor seems to preferentially activate local NO3- signaling (Krouk et al. 2006) and accordingly, did not interfere with Lpr in our experiments. Mechanisms of aquaporin regulation In wild-type Arabidopsis (Col-0), NO3- availability interfered with Lpr without altering PIP transcript and protein abundance. Since PIPs contribute to > 70% of Col-0 Lpr (Sutka et al. 2011), they are likely the main target of NO3-. Whereas a marked 16


role, if any, in governing Lpr.

reduction of PIP2 proteins was observed after 6 d of NO3- starvation in a previous study (Di Pietro et al. 2013), the use of a shorter starvation treatment (3 d) may explain why this reduction was not observed in the present study. Here, we speculate that the control of aquaporin function was essentially post-translational. A dominating

nutritional stress conditions has been described (Di Pietro et al. 2013) but other mechanisms involving protein-protein interactions for instance may be invoked (Maurel et al. 2015). We cannot exclude however that NO3- availability induced slight changes in root anatomy which contributed to Lpr variations. The finding that Lpr of nrt2.1 is tightly linked to PIP expression brings additional support to a general role of aquaporins in NO3-- dependent regulation of Lpr. In this genotype, reduced transcript abundance of a few PIP genes including PIP1;1, PIP1;2, PIP2;1, PIP2;3 could account for a reduced Lpr, under both ST and LN conditions. As a consequence, nitrate deprivation (LN conditions) resulted in a lower abundance of both PIP1s and PIP2s in nrt2.1 compared to Col-0. We realize that in these conditions internal NO3- was dramatically lower in nrt2.1 than in Col-0 and therefore non-saturating effects of NO3- on PIP expression may have been revealed in the former genotype. We note, however, that shoot and root NO3- contents were comparable in the two genotypes under ST conditions, whereas PIP1;1, PIP1;2, PIP2;1, PIP2;3 transcripts levels as well as PIP1s abundance were lower in nrt2.1 mutants than in Col-0. Thus, similar to its overall effects on Lpr, NRT2.1 seems to

17


role of aquaporin phosphorylation in governing Lpr under various abiotic and

govern aquaporin expression, independently of tissue NO3- accumulation, at least in these conditions.

In summary, our work has revealed that the modes of PIP regulation are distinct in Col-0 and nrt2.1. It indicates how plant NO3- and NRT2.1 trigger complex

N-dependent Lpr regulation. NRT2.1 was previously shown to act as a NO3sensor/transducer (Little et al. 2005; Remans et al. 2006a) and the possibility exists that it determines (reduces) the sensitivity of aquaporin gene expression to NO3-, by signaling mechanisms that remain to be determined. Recent studies in rice show that NO3- enhances the amplitude of diurnal variation of aquaporin gene expression (Ishikawa-Sakurai et al. 2014). Thus, the interplay between NO3- and/or NRT2.1 and the circadian clock may also have to be considered. Conservation or divergence of NO3- responses between species It is of note that, in some species, such as Lotus japonicus (Prosser et al. 2006) or sunflower (Gloser et al. 2007), the kinetic effects of N availability (deprivation and resupply) on Lpr.are much faster (~2h) than in Arabidopsis (days). In addition, the conclusion that long-distance N signaling dominates Lpr regulation in Arabidopsis does not preclude that local adaptations to NO3- availability occur in other plant species. For instance, split-root experiments revealed how localized supply of NO3enhances root water uptake and regulates 2 PIP genes, in cucumber and rice, respectively (Gorska et al. 2008a; Ishikawa-Sakurai et al. 2014). These responses may favor N acquisition from NO3- enriched patches whereas a global control by the N 18


transcriptional and post-transcriptional regulations of aquaporins, leading to

status plant (i.e. shoot NO3- accumulation) may help adjust Lpr to the growth capacity of the plant. As suggested in rice (Ishikawa-Sakurai et al. 2014), these two types of mechanisms may potentially co-exist in each plant species to provide the optimal adaptive response to N availability. Integrating the multiple effects of nutrient

challenge in plant research and for crop improvement.

19


availability, in the soil and within the plant body, on water relations represents a major

Materials and methods Plant materials and culture conditions The Arabidopsis thaliana nrt1.1-1 (chl1-5), nrt1.5-1 (SALK_005099), nrt2.1-1 (SALK_008253), nrt2.1-2 (SALK_034529) and nia (G’4-3) genotypes are in Col-0

were described elsewhere (Hu et al. 2009; Li et al. 2007; Lin et al. 2008; Muños et al. 2004; Remans et al. 2006b). Seeds were surface-sterilized, kept for 2 d at 4°C, and grown in clear polystyrene culture plates at 22°C in the light for 10 d, as described (Javot et al. 2003). Seedlings were then transferred to a hydroponic solution [1 mM KNO3, 0.5 mM MgSO4, 2 mM Ca(NO3)2,0.5 mM CaCl2, 0.5 mM NaH2PO4, 50 µM FeEDTA, 50 µM H3BO3, 12 µM MnSO4, 0.70 µM CuSO4, 1 µM ZnSO4, 0.24 µM MoO4Na2, and 100 µM Na2SiO3]. In standard conditions, NO3- was supplied at a 5 mM concentration. To assay the effects of different NO3- concentrations, the standard solution was complemented with 5 mM KNO3- to yield a 10 mM NO3- concentration. A hydroponic solution with 0.5mM NO3- was obtained by replacing 1 mM KNO3 and 1.75 mM Ca(NO3)2 with 1 mM KCl, and 0.75 mM CaCl2 and 1 mM CaSO4, respectively. For a NO3- free solution, 1 mM KNO3 and 2 mM Ca(NO3)2 were replaced with 1 mM KCl, and 1.25 mM CaCl2 and 1.25 mM CaSO4, respectively. Cultures were maintained at a relative humidity of 70% with a 16-h-light at 22°C/8-h-dark at 21°C cycle, and the culture medium was replaced weekly. Root NO3- uptake activity, Lpr measurements and RNA and protein extractions were performed on 21-d-old plants, i.e. at 11 d after transfer in hydroponic solution. 20


background whereas nrt1.2-1 is in Wassilewskija (WS) background. All genotypes

Tissue sampling for RNA and protein extractions was performed at a well-defined time of the day (around 3 pm).To study the effects of external NO3-, the hydroponic solution was replaced as described above at 8 d after transfer and the plants were further grown for 3 d.

The Lpr measurement was performed as described (Boursiac et al. 2005). Briefly, the root system of a freshly detopped plant was inserted into a pressure chamber filled with the hydroponic solution. Pressure was then slowly applied to the chamber, and the rate of sap flow (Jv) exuded from the sectioned hypocotyl was determined by a micro flow-meter at 3 pressures (P) between 0.16 and 0.32 MPa. The Lpr of an individual root system (in mL g-1h-1MPa-1) was calculated from the slope of a plot Jv versus P, divided by the dry weight of the root. Root nitrate uptake activity assay All experiments were performed according to (Remans et al. 2006b). Briefly, the plants were transferred to a 5-cm-diameter petri dish, with the roots (but not the shoot) bathing in a 0.1 mM CaSO4 solution. After 1 min, the solution was replaced with 5 mM or 0.5 mM 15NO3- solution for HATS+LATS or HATS assays, respectively. Plant roots were bathed in these solutions for 5 min, and rinsed for 1 min with 0.1 mM CaSO4 before being harvested, dried at 70°C for 48 h, and weighed. The total amount of

15

N

in dried root samples was measured using an integrated system for continuous flow isotope ratio mass spectrometry. Nitrate uptake activity was calculated as mmole 15

NO3- h-1 g-1 root dry weight. 21


Measurement of Lpr in the pressurized chamber

Nitrate accumulation in shoots and roots Nitrate accumulation in shoots and roots was determined using a nitrate reductase method (Campbell et al. 2006). To release tissue NO3-, whole shoots or roots were dried and incubated in 1.8 mL 0.1 M HCl for 24 h. Root and shoot samples were

aliquote. The principle is that NO3- is reduced to nitrite (NO2-) by nitrate reductase in the

presence

of

NADPH.

Nitrite

subsequently

reacts

with

N-(1-naphthyl)-ethylenediamine dihydrochloride (NNEDD) and sulphanilamide to produce p-sulfobenzene-azo α-naphtylamine, a water soluble red azo dye with a specific absorption peak at 540 nm. The NO3- concentration was determined using calibrated standard solutions and a Victor plate reader (Perkin-Elmer). Total RNA extraction and q-PCR Total RNA was extracted from roots using a SV-RNA isolation kit (Promega) and treated by RQ1 DNase. Poly(dT) cDNA was prepared from 1 µg total RNA using the Transcriptor first-strand cDNA synthesis kit (Roche Life Science). Q-PCR was performed using SYBR Green Sensimix (Quantace) on a Roche LightCycler 480 apparatus. PCR was carried out in 384-well optical reaction plates heated for 10 min to 95°C, followed by 60 cycles with 5 s at 95°C, 8 s at 62°C, 10 s at 72°C, and temperature transitions of 20°C/s. Target quantifications were performed with the specific primer pairs described previously (Postaire et al. 2010), using a set of preselected reference genes [UBQ10 (At4g05320), TIP41-like (At4g34270), F-box family protein (At5g15710), ACT7 (At5g09810), EF-1-a (At5g60390), GAPCP-1 22


diluted 15- and 60-fold, respectively, and NO3- analysis was performed on a 10 µL

(At1g79530), SAND family protein (SFP; At2g28390)] and geNORM analysis methods (Vandesompele et al. 2002). ACT7 and GAPCP-1 were found to be the most stable reference genes between genotypes or after NO3- treatments. ELISAs

2003). Briefly, serial two-fold dilutions in a carbonate buffer (30 mM Na2CO3, 60 mM NaHCO3, pH 9.5) of 0.5 µg of membrane extracts were loaded in triplicate on immunoplates (Maxisorp). An anti-PIP1 antibody raised against 4 out of 5 PIP1s (PIP1;1, PIP1;2, PIP1;3 and PIP1;4) and an anti-PIP2 antibody raised against 3 out of 8 PIP2s (PIP2;1, PIP2;2 and PIP2;3) were used at a 1:2000 dilution (Santoni et al. 2003). Statistical analyses Statistical analyses were conducted using Statistica and GraphPad Prism 6 softwares. The effects of genotype and/or growth conditions were tested using one-way ANOVA and posthoc multiple comparisons were run using a Tukey test. Correlations were investigated using simple or multiple linear regression or Spearman’s rank analysis.

Funding This work was supported by a Federative Project (Rhizopolis) of the Agropolis Fondation (Montpellier, France).

Acknowledgements

23


ELISAs were performed as described previously (Peret et al. 2012; Santoni et al.

Several of our colleagues at the department of Biochimie et Physiologie Moléculaire des Plantes were helpful during this work. Seminal discussions with Dr Hervé Sentenac and his contribution in initiating the project are fully acknowledged. We thank Dr. Sandrine Ruffel and Dr. Philippe Nacry for helpful discussions and gift of

also thank Dr Isabelle Chérel and Prof. Ji-Ming Gong (Shanghai Institute for Biological Sciences, Chinese Academy of Science) for providing complementary seed stocks that were used in preliminary experiments.

24


mutant seed stocks, and Dr. Christelle Taochy for help in NO3- content analysis. We

References Anderberg, H.I., Danielson, J.A. and Johanson, U. (2011) Algal MIPs, high diversity and conserved motifs. BMC Evol. Biol. 11: 110. Anderberg, H.I., Kjellbom, P. and Johanson, U. (2012) Annotation of Selaginella

terrestrial plants. Front. Plant Sci. 3: 33. Barthes, L., Deléens, E., Bousser, A., Hoarau, J. and Prioul, J.-L. (1996) Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools. J. Exp. Bot. 47: 485-495. Bouguyon, E., Brun, F., Meynard, D., Kubeš, M., Pervent, M., Leran, S., et al. (2015) Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1. 1. Nat. Plants 1: 15015. Bouguyon, E., Gojon, A. and Nacry, P. (2012) Nitrate sensing and signaling in plants. Semin. Cell Dev. Biol. 23: 648-654. Boursiac, Y., Chen, S., Luu, D.T., Sorieul, M., van den Dries, N. and Maurel, C. (2005) Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant Physiol. 139: 790-805. Campbell, W.H., Song, P. and Barbier, G.G. (2006) Nitrate reductase for nitrate analysis in water. Environ. Chem. Lett. 4: 69-73. Carvajal, M., Cooke, D.T. and Clarkson, D.T. (1996) Responses of wheat plants to nutrient deprivation may involve the regulation of water-channel function. Planta 25


moellendorffii major intrinsic proteins and the evolution of the protein family in

199: 372-381. Chaumont, F., Barrieu, F., Wojcik, E., Chrispeels, M.J. and Jung, R. (2001) Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiol. 125: 1206-1215.

et al. (2000) Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress. J. Exp. Bot. 51: 61-70. Cramer, M.D., Hawkins, H.-J. and Verboom, G.A. (2009) The importance of nutritional regulation of plant water flux. Oecologia 161: 15-24. Di Pietro, M., Vialaret, J., Li, G.W., Hem, S., Prado, K., Rossignol, M., et al. (2013) Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots. Mol. Cell. Proteomics 12: 3886-3897. Filleur, S., Dorbe, M.-F., Cerezo, M., Orsel, M., Granier, F., Gojon, A., et al. (2001) An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake. FEBS Lett. 489: 220-224. Forde, B.G. (2002) Local and long-range signaling pathways regulating plant responses to nitrate. Annu. Rev. Plant Biol. 53: 203-224. Gloser, V., Zwieniecki, M.A., Orians, C.M. and Holbrook, N.M. (2007) Dynamic changes in root hydraulic properties in response to nitrate availability. J. Exp. Bot. 58: 2409-2415. Gojon, A., Krouk, G., Perrine-Walker, F. and Laugier, E. (2011) Nitrate transceptor(s) in plants. J. Exp. Bot. 62: 2299-2308. 26


Clarkson, D.T., Carvajal, M., Henzler, T., Waterhouse, R.N., Smyth, A.J., Cooke, D.T.,

Gojon, A., Nacry, P. and Davidian, J.-C. (2009) Root uptake regulation: a central process for NPS homeostasis in plants. Curr. Opin. Plant Biol. 12: 328-338. Gorska, A., Ye, Q., Holbrook, N.M. and Zwieniecki, M.A. (2008a) Nitrate control of root hydraulic properties in plants: translating local information to whole plant

Gorska, A., Zwieniecka, A., Holbrook, N.M. and Zwieniecki, M.A. (2008b) Nitrate induction of root hydraulic conductivity in maize is not correlated with aquaporin expression. Planta 228: 989-998. Guo, S., Kaldenhoff, R., Uehlein, N., Sattelmacher, B. and Brueck, H. (2007) Relationship

between

water

and

nitrogen

uptake

in

nitrate-

and

ammonium-supplied Phaseolus vulgaris L. plants. J. Plant Nutr. Soil Sci. 170: 73–80. Hu, H.C., Wang, Y.Y. and Tsay, Y.F. (2009) AtCIPK8, a CBL-interacting protein kinase, regulates the low-affinity phase of the primary nitrate response. Plant J. 57: 264-278. Huang, N.-C., Liu, K.-H., Lo, H.-J. and Tsay, Y.-F. (1999) Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell 11: 1381-1392. Ishikawa-Sakurai, J., Hayashi, H. and Murai-Hatano, M. (2014) Nitrogen availability affects hydraulic conductivity of rice roots, possibly through changes in aquaporin gene expression. Plant Soil 379: 289-300. Javot, H., Lauvergeat, V., Santoni, V., Martin-Laurent, F., Güçlü, J., Vinh, J., et al. 27


response. Plant Physiol. 148: 1159-1167.

(2003) Role of a single aquaporin isoform in root water uptake. Plant Cell 15: 509-522. Johanson, U., Karlsson, M., Johansson, I., Gustavsson, S., Sjövall, S., Fraysse, L., et al. (2001) The complete set of genes encoding major intrinsic proteins in

proteins in plants. Plant Physiol. 126: 1358-1369. Krapp, A., David, L.C., Chardin, C., Girin, T., Marmagne, A., Leprince, A.-S., et al. (2014) Nitrate transport and signalling in Arabidopsis. J. Exp. Bot. 65: 789-798. Krouk, G., Lacombe, B., Bielach, A., Perrine-Walker, F., Malinska, K., Mounier, E., et al. (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev. Cell 18: 927-937. Krouk, G., Ruffel, S., Gutierrez, R.A., Gojon, A., Crawford, N.M., Coruzzi, G.M., et al. (2011) A framework integrating plant growth with hormones and nutrients. Trends Plant Sci. 16: 178-182. Krouk, G., Tillard, P. and Gojon, A. (2006) Regulation of the high-affinity NO3− uptake system by NRT1. 1-mediated NO3− demand signaling in Arabidopsis. Plant Physiol. 142: 1075-1086. Léran, S., Varala, K., Boyer, J.-C., Chiurazzi, M., Crawford, N., Daniel-Vedele, F., et al. (2014) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 19: 5-9. Laur, J. and Hacke, U.G. (2013) Transpirational demand affects aquaporin expression in poplar roots. J. Exp. Bot. 64: 2283-2293. 28


Arabidopsis provides a framework for a new nomenclature for major intrinsic

Lezhneva, L., Kiba, T., Feria-Bourrellier, A.-B., Lafouge, F., Boutet-Mercey, S., Zoufan, P., et al. (2014) The Arabidopsis nitrate transporter NRT2. 5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants. Plant J. 80: 230-241.

Biochim. Biophys. Acta 1840: 1574-1582. Li, W., Wang, Y., Okamoto, M., Crawford, N.M., Siddiqi, M.Y. and Glass, A.D. (2007) Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiol. 143: 425-433. Lin, S.H., Kuo, H.F., Canivenc, G., Lin, C.S., Lepetit, M., Hsu, P.K., et al. (2008) Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20: 2514-2528. Little, D.Y., Rao, H., Oliva, S., Daniel-Vedele, F., Krapp, A. and Malamy, J.E. (2005) The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. Proc. Natl. Acad. Sci. USA 102: 13693-13698. Liu, J., Equiza, M.A., Navarro-Rodenas, A., Lee, S.H. and Zwiazek, J.J. (2014) Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins. Planta 240: 553-564. Lopez, D., Bronner, G., Brunel, N., Auguin, D., Bourgerie, S., Brignolas, F., et al. (2012) Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation. J. Exp. Bot. 63: 2217-2230. 29


Li, G., Santoni, V. and Maurel, C. (2014) Plant aquaporins: roles in plant physiology.

Marchive, C., Roudier, F., Castaings, L., Brehaut, V., Blondet, E., Colot, V., et al. (2013) Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nat. Comm. 4: 1713. Maurel, C., Boursiac, Y., Luu, D.-T., Santoni, V., Shahzad, Z. and Verdoucq, L. (2015)

Muños, S., Cazettes, C., Fizames, C., Gaymard, F., Tillard, P., Lepetit, M., et al. (2004) Transcript profiling in the chl1-5 mutant of Arabidopsis reveals a role of the nitrate transporter NRT1. 1 in the regulation of another nitrate transporter, NRT2. 1. Plant Cell 16: 2433-2447. Nacry, P., Bouguyon, E. and Gojon, A. (2013) Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource. Plant Soil 370: 1-29. Peret, B., Li, G., Zhao, J., Band, L.R., Voss, U., Postaire, O., et al. (2012) Auxin regulates aquaporin function to facilitate lateral root emergence. Nat. Cell Biol. 14: 991-998. Postaire, O., Tournaire-Roux, C., Grondin, A., Boursiac, Y., Morillon, R., Schaffner, A.R., et al. (2010) A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis. Plant Physiol. 152: 1418-1430. Prosser, I.M., Massonneau, A., Smyth, A.J., Waterhouse, R.N., Forde, B.G. and Clarkson, D.T. (2006) Nitrate assimilation in the forage legume Lotus japonicus L. Planta 223: 821-834. 30


Aquaporins in plants. Physiol. Rev. 95: 1321-1358.

Radin, J.W. and Boyer, J.S. (1982) Control of leaf expansion by nitrogen nutrition in sunflower plants: role of hydraulic conductivity and turgor. Plant Physiol. 69: 771-775. Remans, T., Nacry, P., Pervent, M., Filleur, S., Diatloff, E., Mounier, E., et al. (2006a)

triggering root colonization of nitrate-rich patches. Proc. Natl. Acad. Sci. USA 103: 19206-19211. Remans, T., Nacry, P., Pervent, M., Girin, T., Tillard, P., Lepetit, M., et al. (2006b) A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiol. 140: 909-921. Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. and Maeshima, M. (2005) Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant Cell Physiol. 46: 1568-1577. Santoni, V., Vinh, J., Pflieger, D., Sommerer, N. and Maurel, C. (2003) A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots. Biochem. J. 373: 289-296. Stitt, M. (1999) Nitrate regulation of metabolism and growth. Curr. Opin. Plant Biol. 2: 178-186. Sutka, M., Li, G., Boudet, J., Boursiac, Y., Doumas, P. and Maurel, C. (2011) Natural variation of root hydraulics in Arabidopsis grown in normal and salt-stressed conditions. Plant Physiol. 155: 1264-1276. 31


The Arabidopsis NRT1.1 transporter participates in the signaling pathway

Tournaire-Roux, C., Sutka, M., Javot, H., Gout, E., Gerbeau, P., Luu, D.T., et al. (2003) Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature 425: 393-397. Unkles, S.E., Wang, R., Wang, Y., Glass, A.D., Crawford, N.M. and Kinghorn, J.R.

plant cells. J. Biol. Chem. 279: 28182-28186. Vandeleur, R.K., Sullivan, W., Athman, A., Jordans, C., Gilliham, M., Kaiser, B.N., et al. (2014) Rapid shoot-to-root signalling regulates root hydraulic conductance via aquaporins. Plant, Cell Environ. 37: 520-538. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: RESEARCH0034. Wang, Y.-H., Garvin, D.F. and Kochian, L.V. (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiol. 127: 345-359. Wang, Y.Y., Hsu, P.K. and Tsay, Y.F. (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci. 17: 458-467. Zhang, H., Jennings, A., Barlow, P.W. and Forde, B.G. (1999) Dual pathways for regulation of root branching by nitrate. Proc. Natl. Acad. Sci. USA 96: 6529-6534. Zheng, D., Han, X., An, Y., Guo, H., Xia, X. and Yin, W. (2013) The nitrate transporter 32


(2004) Nitrate reductase activity is required for nitrate uptake into fungal but not

NRT2.1 functions in the ethylene response to nitrate deficiency in Arabidopsis. Plant Cell Environ. 36: 1328-1337. Zhuo, D., Okamoto, M., Vidmar, J.J. and Glass, A.D. (1999) Regulation of a putative high‐affinity nitrate transporter (Nrt2; 1At) in roots of Arabidopsis thaliana. Plant


J. 17: 563-568.

33

Figure legends Fig. 1. Root hydraulic conductivity (Lpr) from wild-type (Col-0, WS), nrt1.1-1, nrt1.2-1, nrt1.5-1, nrt2.1-1 and nia plants in standard conditions. All mutants, except nrt1.2-1 which was identified in a WS background, derive from Col-0 plants. Lpr was measured

The values are relative means (in % ± SE), which respect to Lpr in corresponding wild-type plants. Data (from the indicated number of plants, n, in brackets) were

pooled from 5 (Col-0, nrt1.1-1, nrt1.5-1, nrt2.1-1), 3 (nia) or 2 (WS, nrt1.2-1) independent plant cultures. One-way ANOVA with Tukey’s multiple test indicates a statistically difference between nrt2.1-1 and others (p < 0.0001).

Fig. 2. Effects of external NO3- on Lpr and NO3- content in shoots and roots. Plants of the indicated genotypes were successively grown for 18 d in ST conditions and for 3 d in the presence of the indicated external NO3- concentration, prior to measurements of Lpr (■) and NO3- content in shoots (▲) and roots (♦). The data were pooled from at least 2 independent plant cultures with n > 10 plants. The values are means ± SE. Lpr data from plants grown at 5 mM NO3 were included in the analysis shown in Fig.1.

Fig. 3. Lpr of nrt2.1 in ST and LN conditions. Wild-type (Col-0) and mutant (nrt2.1-1, nrt2.1-2) plants were exposed to 0.5 mM (LN) or 5 mM (ST) external NO3- as exemplified in Fig. 2. All Lpr values were normalized to that of Col-0 in ST conditions (119.2 ml g-1 h-1 MPa-1). Data were pooled from at least 3 independent plant cultures 34


using the pressure chamber technique, as described in the Materials and methods.

and the indicated numbers of plants. Values are means ± SE, and letters indicate statistically different values at p < 0.05 (ANOVA).

Fig. 4. Correlation between Lpr and NO3- content in shoots and roots. Plants of the

grown in the presence of 0, 0.5 or 5 mM external NO3-. The figure shows plots of Lpr against NO3- content in shoots (A) and roots (B). The correlation coefficient (R2) and p value corresponding to the regression lines are indicated.

Fig. 5. Effects of external NO3- on PIP transcript abundance in Col-0 and nrt2.1 plants. The expression of all 13 PIP was characterized using q-PCR in roots of the indicated genotypes grown under ST or LN conditions. Data from selected PIPs including the four most highly expressed ones (PIP1;1, PIP1;2, PIP2;1, PIP2;2) are shown (See complementary data in Supplemental Fig. S5). For each gene, transcript abundance (mean ± SE) from 3 independent plant cultures was normalized to transcript abundance in Col-0 grown in ST conditions.

Fig. 6. Correlation between Lpr and PIP transcript abundance in roots of Col-0 grown in ST conditions (see asterisks, *) and nrt2.1-1 and nrt2.1-2 grown in ST and LN conditions. The figures show plots of relative Lpr (normalized to Lpr of Col-0 in ST conditions) against relative transcript abundance of the indicated gene (normalized to transcript abundance of Col-0 in ST conditions). The parameters (R2 and p values) of 35


indicated genotypes (Col-0, ●; nrt1.1-1, ▲; nrt1.5-1, +; nrt2.1-1, ○; nia, ■) were

each linear regression are indicated.

Fig. 7. Abundance of PIP1s and PIP2s and their correlation with Lpr in Col-0, nrt2.1-1 and nrt2.1-2 plants grown under ST or LN conditions. ELISAs using anti-PIP1 (A) or

plant cultures. The pooled data were normalized to PIP1 or PIP2 abundance in Col-0 grown in ST conditions. Lpr was normalized to Lpr of Col-0 in ST conditions and plotted against relative PIP1 (C) and PIP2 (D) abundance. The dots corresponding to Col-0 in ST conditions are indicated with an asterisk (*). The parameters (R2, p) of each linear regression are indicated.

36


anti-PIP2 (B) antibodies were performed in root samples from at least 3 independent

a

Figure 1

Li et al., 2015

(14)

a

a

n r t1 .2 - 1

(17)

WS

a

(32)

n ia

a

(35)

n r t2 .1 - 1

(35)

n r t1 .5 - 1

(36)

n r t1 .1 - 1

C o l-0

R e la tiv e L p r (% ) 120

(14)

a

90

b

60

30

0

1.2

90 0.8 60 0.4

30

0

0.5

5

10

nrt1.1-1

2.0 1.6

90

1.2 60 0.8 30

0.4

0

160

0

0.5

5

10

200 160

1.2 80 0.6

40

0.0

0

2.4

120

0.0 0

0.5

5

10

2.0

120

1.8

80

1.2

40

0.6

0

0.0 0.5

5

Lpr (ml/h/g/MPa)

nrt2.1-1 NO3- (mmol/gDW)

Lpr (ml/h/g/MPa)

1.8

120

nia

0

2.4

nrt1.5-1

1.6

90

1.2 60 0.8 30

10

0.4

0

0.0 0

0.5

5

10

NO3- concentration (mM)

NO3- concentration (mM)

Figure 2

Li et al., 2015

NO3- (mmol/gDW)

Lpr (ml/h/g/MPa)

120

0.0

NO3- (mmol/gDW)

0

Lpr Root NO3Shoot NO3-

Lpr (ml/h/g/MPa)

Lpr (ml/h/g/MPa)

120

NO3- (mmol/gDW)

1.6

Col-0

NO3- (mmol/gDW)

150

120

(36) (36) a

Relative L pr (%)

100 b

ST

(32) (25) b

80

c

LN

(22) (20) b c

60 40 20 0 Col-0

nrt2.1-1

Figure 3

Li et al., 2015

nrt2.1-2

A

B

Figure 4

Li et al., 2015

a a a

a a

a

ab

ab

a ab

ab

b

b ab

c

bc

c

c

c

a a a a

a

a a

ab

b b

ab

b

b

ab bc

b

b c

Figure 5

Li et al., 2015

c

b

Figure 6

Li et al., 2015

Figure 7

Li et al., 2015

Molecular Mechanism Underlying the Plant NRT1.1 Dual-Affinity Nitrate Transporter.

Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development.

AtNPF5.5, a nitrate transporter affecting nitrogen accumulation in Arabidopsis embryo.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana.

Signal interactions in the regulation of root nitrate uptake.

Transporters Involved in Root Nitrate Uptake and Sensing by Arabidopsis.

Environmental Nitrate Stimulates Abscisic Acid Accumulation in Arabidopsis Root Tips by Releasing It from Inactive Stores.

Nitrate and periplasmic nitrate reductases.

Nitrate Storage and Dissimilatory Nitrate Reduction by Eukaryotic Microbes.

Nitrate and nitrite reduction by liquid membrane-encapsulated whole cells.

Post-flowering nitrate uptake in wheat is controlled by N status at flowering, with a putative major role of root nitrate transporter NRT2.1.

Regulation of transcript level and synthesis of nitrate reductase by phytochrome and nitrate in turions of Spirodela polyrhiza (L.) Schleiden.

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa.

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling.

MADS-box transcription factor OsMADS25 regulates root development through affection of nitrate accumulation in rice.

The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress.

NRT1.5 is involved in lateral root development under potassium deprivation.

Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity.

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots, plantlets and seedlings of Pinus pinaster.

Regulation of spinach-leaf nitrate reductase by reversible phosphorylation.

Ammonium reduces oxalate accumulation in different spinach (Spinacia oleracea L.) genotypes by inhibiting root uptake of nitrate.

The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants.

Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins.