Calcium signaling networks mediate nitrate sensing and responses in Arabidopsis

ABSTRACT

Nitrate signaling integrates and coordinates the expression of a wide range of genes, metabolic pathways and ultimately, plant growth and development. Calcium signaling is proved to be involved in the primary nitrate response pathway. However, it is much less understood how calcium signaling mediates nitrate sensing and responses from the extracellular space to cytoplasm, then to the nucleus. In this review, we describe how transceptor-channel complex (cyclic nucleotide-gated channel protein 15 interacting with nitrate transceptor, CNGC15-NRT1.1), calcineurin B-like proteins (CBLs, CBL1, CBL9), CBL-interacting protein kinases (CIPKs), phospholipase C (PLC) and calcium-dependent protein kinases (CDPKs, also CPKs), acting as key players, complete a potential backbone of the nitrate-signaling pathway, from the plasma membrane to the nucleus. NRT1.1 together with CBL1/9-CIPK23 and CBL-CIPK8 links the NO₃⁻ signaling to cytoplasmic and nuclear regulators and triggers downstream NO₃⁻ responses. PLCs and inositol 1, 4, 5-triphosphate (IP3) connect NO₃⁻ signaling and cytoplasmic Ca²⁺ signature. CPK10/30/32 fill the gap between NRT1.1 and NIN-like protein (NLP) transcription factors. The arabidopsis nitrate regulated1 (ANR1) is induced from the endosome by the Ca²⁺–CPKs–NLPs signaling pathway activated by the unphosphorylated form of NRT1.1 (NRT1.1 ^T101A) at high nitrate condition. Understanding how calcium signaling interconnects the upstream nitrate sensor complex with downstream multiple sensors of the nitrate-signaling pathway is key to completing the nutrient–growth regulatory networks.

KEYWORDS:

• Calcium signaling
• nitrate
• CNGC15-NRT1.1
• CBLs
• CIPKs
• PLCs
• CPKs
• ANR1
• primary nitrate response

1. Introduction

Nitrate (NO₃^–) in the soil is not only an essential source of nitrogen for plants, but also functions as a signaling molecule, modulating gene expression and a multitude of physiological and developmental processes, ranging from the control of plant growth, root development, leaf development, seed dormancy, and flowering time as well as the transport and assimilation of NO₃.^−1–7 Plant adaptation to NO₃ ^– availability is accompanied by a massive and rapid reprogramming of the transcriptome, defined this multifaceted molecular phenomena as the primary nitrate response (PNR). The NO₃ ^– sensing machinery consists of a dual-affinity NO₃⁻ transporter and sensor (defines this protein as a “transceptor”), named NRT1.1,^6,8,9 nitrate-responsive transcripts, protein phosphatase 2 C (PP2C) and transcription factors.^10–18 Despite the progress represented by the identification of the elements of the NO₃⁻ -sensing machinery, the molecular regulatory mechanism involving the primary nitrate signaling upstream and the multiple factors and sensors downstream remain to be ascertained. Calcium functions as a key second messenger in diverse physiological and developmental pathways, being associated with stress response, self-incompatibility, nodulation, and the regulation of ion transport.^19–30 Ca²⁺ is also involved in NO₃ ^– signaling, which was first described 20 years ago in maize and barley.^31,32 Riveras et al.³³ revealed a transient cytoplasmic Ca²⁺ increase induced by NO₃⁻ in Arabidopsis, which was inhibited by the Ca²⁺ channel blocker lanthanum chloride (LaCl₃). Furthermore, this cytoplasmic Ca²⁺ increase was abolished in the NRT1.1 mutants chl1-5 and chl1-9, pointing to a functional transceptor being indispensable for this response. In addition, it has been shown that inositol 1, 4, 5-triphosphate (IP3) concentrations increased in response to NO₃ ^– treatments, indicating that PLC activity also takes part in this process.³³ Notably, the calcium-dependent kinases calcineurin B-like protein (CBL)-interacting protein kinases (CIPKs) and calcium-dependent protein kinases (CPKs) are the master molecular actors regulating the NO₃^–-signaling pathway. The primary nitrate response pathway has a primary backbone linking the nitrate sensor NRT1.1, CPK calcium-dependent kinases and NLP7-induced gene expression except for the mechanism underlying Ca²⁺ influx. Ca²⁺ enters the cytoplasm through Ca²⁺ channels, which are located in cell membranes and organelles, such as vacuoles and endoplasmic reticulum. Cyclic nucleotide gated channel (CNGCS), denoted as Ca²⁺ -permeable channels, have been extensively researched in a variety of Ca²⁺-signaling processes in plants.^34–36 In this review, we discuss how CNGC15-NRT1.1, CBLs, CIPKs, PLCs and CPKs preliminarily link the upstream and downstream NO₃ ^– sensing signaling pathway, and we highlight the importance of molecular and genetic analyses in the identification of novel features of calcium in the NO₃ ^– signaling pathway.

2. CNGC15-NRT1.1 couples NO₃⁻ sensing to Ca²⁺ signaling

Cyclic nucleotide gated channel (CNGCS) is a class of nonselective cationic channels with permeability to monovalent and divalent cations, consisting of 20 members in Arabidopsis, which is one of the most important proteins in plants mediate Ca²⁺-signaling processes.^34–36 The membrane-located nitrate transceptor NRT1.1 has been identified as a central component for primary nitrate responses in Arabidopsis.^37,38 Interestingly, Wang et al.³⁹ discovered a nutrient transceptor-channel complex named CNGC15-NRT1.1 as a molecular switch controls the channel activity and Ca²⁺ influx in a nitrate-dependent manner. CNGC15 mutants exhibited the characteristic loss of NO₃⁻ -induced Ca²⁺ signature, which is similar to that in NRT1.1-deficient mutants chl1-5. CNGC15 and NRT1.1 formed a plasma membrane Ca²⁺-permeable channel, but the channel activity of the heterocomplex was inhibited. After addition of NO₃⁻, the interaction between CNGC15-NRT1.1 was weakened, and the Ca²⁺ channel activity of CNGC15-NRT1.1 complex was restored, thus forming the CNGC15-NRT1.1 complex which could be dynamically gated by NO₃⁻. This novel “transceptor-channel” or “transporter-calcium channel” gated by small nutrient molecules further improves the nitrate nutrient signal transduction pathway and nitrate triggered calcium signaling transduction pathway in plants.

3. CBL-CIPKs in NO₃⁻ signaling

Calcineurin-B like proteins (CBL) is a group of calcium sensor proteins that interact with CIPK kinases (CBL-interacting protein kinases), which phosphorylate their downstream target proteins.^40,41 In Arabidopsis, 10 calcineurin B-like proteins (CBLs), with 26 CIPKs have been discovered so far.^41–43 CBL7 was reported to regulate the low-concentration nitrate response process, which was mainly expressed in the roots of seedlings and induced by nitrate starvation. In the absence of nitrate, the loss of CBL7 inhibited root growth and decreased the expression of NRT2.4/2.5.⁴⁴ In addition, CBL1 and ABI2, a member of the protein phosphatase 2 C family, were found to regulate nitrate transport, sensing, and signaling pathways.^10,45 In particular, the NO₃ ^– signaling needs to be transmitted to the nucleus and magnified through cytoplasmic regulators when NO₃ ^– is sensed by NRT1.1.⁴⁶ The CBL-CIPK system may play a key role in the NO₃^–-signaling pathway, linking up the NO₃⁻ signal with cytoplasmic and nuclear regulators and triggering downstream NO₃⁻ responses.

CBL1 and CBL9 interact with CIPK23 and recruit the kinase to the plasma membrane where the substrate(s) of CIPK23 may reside, regulating the nitrate transporter NPF6.3/NRT1.1 via phosphorylation to modulate nitrate uptake.⁴³ NPF6.3/NRT1.1has been characterized as a dual-affinity NO₃ ^– transporter, with T101-phosphorylated NRT1.1 acting as a high-affinity NO₃ ^– transporter, but as a low-affinity NO₃⁻ transporter when it is dephosphorylated.³⁷ NRT1.1 is a transceptor for NO₃^–, the activity of which regulates a complicated network of multiple kinases and phosphatases affecting PNR.^8,10 The calcium-dependent protein kinase CIPK23 was found to phosphorylate T101 of NRT1.1 when NO₃ ^– concentrations were low^8,47 (Figure 1). This suggested that NRT1.1 may trigger the changes in NO₃^–-induced calcium increases by regulating a dual-affinity binding and a phosphorylation switch in response to a range of NO₃ ^– concentrations (Figure 1).

Figure 1. Calcium signaling networks mediating nitrate signaling pathway. NO₃ ^– is sensed by CHL1/NRT1.1/NPF6.3 under both high and low concentrations. CIPK8 (a CBL-interacting protein kinase, interacting with CBL) regulates NRT1.1 in low-affinity responses, whereas CIPK23 (a CBL-interacting protein kinase, interacting with CBL1/9) phosphorylates NRT1.1 under high-affinity responses. CNGC15-NRT1.1 as a molecular switch controls the channel activity and Ca²⁺ influx. NRT1.1 and PLC activity co-regulate the increase in cytoplasmic Ca²⁺ concentration in response to NO₃^–. When Ca²⁺ accumulates in the nucleus, NLP7 is phosphorylated by CPKs and is retained in the nucleus to regulate PNR. Ca²⁺–CPKs–ANR1 signaling pathway is activated by unphosphorylated form of NRT1.1 (NRT1.1^T101A) at high nitrate (HN) condition

Another CBL-CIPK gene that was elucidated to to regulate NO₃ ^– signaling is CIPK8, especially in low-affinity responses.⁴⁸ Hu et al.⁴⁸ identified that the expression of AtCIPK8 was strongly and rapidly induced by NO₃ ^– (Table 1). Thus, this work described a potential link between calcium signaling and NO₃ ^– signaling. In addition, they confirmed that CIPK8 positively mediated the NO₃ ^– responses of PNR marker genes and modulated primary root growth using cipk8 knockout mutants.⁴⁸ Although a role for AtCIPK8 in regulating NO₃ ^– sensing and signaling has been implicated, many outstanding questions remain to be answered, such as whether NO₃⁻ can trigger Ca²⁺-CIPK signaling, how CIPK8 perceives cytoplasmic calcium increases, and the direct target(s) of CIPK8 remains to be revealed in the NO₃⁻ signaling network.

Table 1. CNGC, CBLs, CIPKs, CPKs, NLP7 and ANR1 involved in nitrate signaling

Gene name	Gene locus	Function	Transcriptional regulation by NO3^−	Reference
AtCNGC15	AT2G28260	forms a molecular switch with NRT1.1 that controls calcium influx depending on nitrate levels	Induced by NO3^−	^Citation39
AtNRT1.1	AT1G12110	a dual-affinity NO3- transporter and sensor	Induced by NO3^−	^Citation6
AtCBL1	AT4G17615	interacts with CIPK23	ND	^Citation43
AtCBL9	AT5G47100	interacts with CIPK23	ND	^Citation43
AtCIPK23	AT1G30270	negatively regulates the primary nitrate response	transiently induced by NO3^−	^Citation8
AtCIPK8	AT4G24400	positively regulates the nitrate-induced expression of primary nitrate response genes	Induced by NO3^−	^Citation48
AtCPK10	AT1G18890	fills the nitrate signaling gap	Induced by NO3^−	^Citation56
AtCPK30	AT1G74740	fills the nitrate signaling gap	Induced by NO3^−	^Citation56
AtCPK32	AT3G57530	fills the nitrate signaling gap	Induced by NO3^−	^Citation56
AtNLP7	AT4G24020	fills the nitrate signaling gap	Induced by NO3^−	^Citation56
AtANR1	AT2G14210	fills the Ca^2+-ANR1 signaling pathway	Induced by NO3^−	^Citation59

4. PLCs in NO₃ ^– signaling

PLCs are key enzymes in phosphatidyl inositol turnover, accompanied by multiple second messengers.⁴⁹ Based on differences in substrate specificities, PLCs can be divided into phosphoinositide-specific phospholipase C (PI-PLC) and nonspecific phospholipase C in plants.⁵⁰ IP3 and diacylglycerol (DAG) are the two second intracellular messengers generated from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzed by PI-PLCs.^50,51 IP3 induces Ca²⁺ released from internal stores (Figure 1). The released Ca²⁺ may be acting as a second messenger in the NO₃⁻ -signaling network. Interestingly, it has been shown that IP3 and cytoplasmic Ca²⁺ levels increased in response to NO₃ ^– treatment, but this response was blocked by the PLC inhibitor U73122.³³ This result suggested that PLC is required for NO₃^–-dependent increases in cytoplasmic Ca²⁺ levels and IP3. Furthermore, it has been shown that an increase in IP3 concentration in Arabidopsis roots in response to NO₃ ^– treatments also requires NRT1.1 for activation of a PLC activity.³³ In addition, DAG can not only activate protein kinase C to transduce the signal to effector molecules but can also generate diacylglycerolpyrophosphate, which serves as a reasonable substrate for phosphatidic acid (PA) under the action of phospholipase D. Nevertheless, it is currently largely unknown whether PA affects cytoplasmic Ca²⁺ concentration in plants.

5. CPKs in NO₃⁻ signaling

5.1. CPK10/30/32 in NO₃⁻ signaling

Ca²⁺-dependent protein kinases (CDPKs, in Arabidopsis designated as CPKs), a family of Ser/Thr kinases, contains a calcium binding site (EF hand) in their C-terminal region. Binding of Ca²⁺ to the EF hand stimulates a conformational change, thus allowing autophosphorylation of the kinase.^42,52 There are 34 CPK genes in the Arabidopsis genome, and more than half of them have been functionally characterized to modulate varying signaling pathways.^41,53–55 CPKs are the long-anticipated molecular link between the major players in the NO₃⁻ signaling pathway, the NO₃⁻ transceptor NRT1.1 located in the plasma membrane and the NIN-like protein (NLP) transcription factors located in the cell nucleus.^46,56 NLPs have been identified as key transcription factors of primary nitrate responses in Arabidopsis. NO₃⁻ can not only induce calcium increase in the nucleus but also trigger Ca²⁺-CPK signaling⁵⁶(Figure 1). Then Liu et al.⁵⁶ identified subgroup III CPKs acting as master regulators mediating primary NO₃⁻ signaling. The development of an integrated seeding and cell-based analysis method, as well as a versatile single-cell analysis system, has contributed to the report of unique Ca²⁺ signaling triggered by nitrate so as to identify initially the mechanism of nitrate signaling transduction. The researchers then demonstrated that CPKs are the master regulators mediating primary NO₃⁻ signaling by using an ultrasensitive calcium biosensor, a sensitized and targeted functional genomic screen, and a chemical switch (a unique mutant system) successfully overcome the embryo lethality of cpk10 cpk30 double mutant. However, it remains unknown how plants transmit the nitrate signaling downstream via calcium-dependent protein kinases–CPK10, CPK30 and CPK32 after sense this signaling. The study reported that nitrate can not only induce Ca²⁺ accumulation in the nucleus, but also can induce CPKs translocation in the nucleus. Furthermore, CPK10 CPK30 and CPK32 phosphorylated the key transcription factor of PNR named NLP7 in the nucleus in response to nitrate. Phosphorylation proteomic analysis showed that the phosphorylation site was Ser205 in NLP7, and NLP7 (Ser205A) mutant lacking the CPK10, CPK30 and CPK32 phosphorylation site was not present in the nucleus and could not complement the root phenotype of the mutant nlp7, indicating that Ca²⁺ acted as a second messenger in response to nitrate and then modulate CPK activity so as to control the entry of NLP7 into the nucleus to interact with NLP7. This findings connected a new Ca²⁺–CPK–NLP signaling cascade to comprehensive nitrate responses and revealed a previously unrecognized function of Ca²⁺-sensor CPKs as master regulators that modulating the primary nitrate-signaling.⁵⁶However, it remains incompletely unclear how and why CPKs are shuttled from the cytoplasm into the nucleus in response to NO₃^–; whether plants can perceive cytoplasmic Ca²⁺ waves as opposed to nuclear ones; and which compartment can provide a link between cytosolic and nuclear calcium concentration.

5.2 Ca²⁺–CPKs–ARABIDOPSIS NITRATE REGULATED1 (ANR1)

ANR1, as a member of the plant MIKC-type MADS box family of transcription factors, was first found to play roles in linking external NO₃⁻ to lateral root (LR) development.⁵⁷ Gan et al.⁵⁸ illustrated that ANR1 positively regulated LR development and early seedling growth, in response to NO₃⁻ regulation in Arabidopsis thaliana. Most recently, Zhang et al. reveals that Ca²⁺–CPKs–ANR1 signaling pathway stimulated from the endosome, in conjunction with auxin transporting, plays an essential role in lateral root growth, which is regulated by the phosphorylated form of NRT1.1.⁵⁹ The NRT1.1 ^T101A nonphosphorylatable mutants exhibited a transient increase of Ca²⁺ signaling in cytoplasm in both high nitrate (HN) and low nitrate (LN) conditions. But the NRT1.1 ^T101D phosphomimetic mutants showed a reduction. This is an interesting point and future efforts are needed to investigate the detailed mechanism. In addition, the ANR1 expression in T101A line was higher than that of T101D line under HN condition. Therefore, the Ca²⁺–CPKs–NLPs signaling pathway could be activated from the endosome by the nonphosphorylated state of NRT1.1 together with elevating the expression of ANR1. Despite this progress, how the phosphorylated and dephosphorylated variants of NRT1.1 at T101 in LN condition affect Ca²⁺ signaling through the ANR1 deserves for further study. It will be interesting to explore the detailed mechanism of how ANR1 links cytoplasmic Ca²⁺ to PNR in the nucleus to better understand how calcium signaling interconnects the upstream nitrate sensor complex with downstream multiple sensors of the nitrate-signaling pathway.

6. Future research directions

This review describes how calcium signaling is involved in the NO₃ ^– -sensing and -signaling pathways so as to establish the potential backbone of the NO₃^–-signaling pathway, from the plasma membrane to the nucleus. A single nitrate ‘transceptor’ NRT1.1 mediates both high-affinity and low-affinity nitrate uptake up to its phosphorylated status at threonine residue 101, regulated by CIPK23. The protein kinase CIPK8 is responsible for NO₃ ^– sensing by regulating the low-affinity phase of the PNR.⁴⁸ Future research should focus on how CIPK8 participates in the NO₃^–-sensing pathway, such as the molecular mechanism elucidating the identity of the elements upstream and downstream of the sensors, to fill the gap in the NO₃^–-sensing network. In addition, the FIP1 (factor interacting with poly (A) polymerase) gene negatively regulated the expression of CIPK8 and CIPK23 when NO₃⁻ was present.⁶⁰ It will be interesting to elucidate how FIP1 regulates co-ordination of the calcium signal with the NO₃ ^– signal, which will complement the NO₃^–-signaling pathway connection with CIPKs. Nevertheless, it is also emerging that we need to decipher the mechanistic details of how precisely CBL-CIPK complexes are formed and how they recognize their targets in much more resolution in order to achieve a more comprehensive understanding of plant nutrition regulation.

NRT1.1 and PLC activity co-regulate the increase in cytoplasmic Ca²⁺ concentration in response to NO₃^–. In this process, PLC hydrolyzes PIP2 into IP3 and DAG, with the increased IP3 concentration correlating positively with an increase in cytoplasmic Ca²⁺ concentration. Future research should focus on determining whether IP3 directly triggers the increase in cytoplasmic Ca²⁺ concentration or operates through its phosphorylated product IP6 in NO₃^–-mediated Ca²⁺ release in the NO₃ ^– PLC-Ca²⁺ pathway.

CPKs bridge the gap between calcium increases triggered by NO₃ ^– and NLP7/6, which are recognized to be pivotal transcription factors of PNR.^46,56 This is the first time that a branch of the NO₃^–-signaling pathway has been revealed in its entirety. This finding prompts important questions such as whether CPKs can also phosphorylate NLP7 in the cytoplasm to regulate its nuclear import and whether NRT1.1 is also regulated by calcium through CIPK or CBL. Although phosphoproteomic analysis has shown that serine 205 of NLP7 is phosphorylated in the presence of NO₃ ^– in the nucleus,⁵⁶ the mechanism behind the change in nuclear calcium concentrations remain to be elucidated. Future work should pay great attention to identifying the receptors, channels and other regulators playing roles in creating complex Ca²⁺ signatures in response to NO₃^–, other nutrients, microRNAs, long non-coding RNAs, circular RNAs, peptides, hormones and environmental cues; this research will be accelerated by new advances in the development of ultrasensitive Ca²⁺ biosensors.

Despite the critical importance of the calcium signals being transmitted downstream by CPKs in plants, the biochemical pathways that underpin the fundamental cellular program connecting [Ca²⁺]_cyt to [Ca²⁺]_nucl remain largely unknown. The changes in intracellular calcium concentration are not a synchronized process, either temporally or spatially.⁶¹ Under different conditions, calcium concentrations in different organelles and other sub-cellular compartments vary, and these different calcium signatures participate in different cellular events. It is well known that the nuclear pore complex (NPC) forms a large channel for the free diffusion of ions and macromolecules.⁶² Calcium could diffuse into and out of the nucleoplasm through the NPC, and changes in the nuclear calcium concentration would be dependent on the cytoplasmic calcium concentration. Researchers in this field urgently require Ca²⁺-signaling researchers to provide a better understanding of the changes in calcium concentration in the cytoplasm and the nucleus in order to fill the NO₃ ^– gap in the NO₃^–-signaling pathway so as to refine our understanding of Ca²⁺-signaling processes in plants.

NO₃ ^– signaling is closely associated with hormonal signaling in order to adjust plant growth and development in response to nutrient availability.^63–65 It is noteworthy that CPK10 and CPK30 were demonstrated to be related to abscisic acid (ABA) signaling 20 years ago.⁶⁶ This exciting link opens up promising avenues of research on the co-control of the NO₃^–- and ABA-signaling pathways. This information can provide us with a greater understanding of the intimate connections between NO₃⁻ -signaling and hormonal signaling pathways. NRT1.1 has been demonstrated to play diverse roles in plants, not only in nitrate uptake and transport, and nitrate signal transduction, but also in auxin transport and mediating root development⁶⁷(Wang et al 2020). AtNRT1.1 has been verified to transport auxin and regulate nitrate-modulated lateral root development (Krouk et al., 2010).⁶⁸ In addition, the phosphorylated form of NRT1.1 (named as NRT1.1^T101D) accelerates auxin flux and inhibits lateral root elongation under low nitrate conditions³⁸ (Bouguyon et al., 2015), however, the unphosphorylated form of NRT1.1 (NRT1.1 ^T101A) suppresses auxin transport and triggers Ca²⁺–CPKs–ANR1 signaling pathway to modulate LR development⁵⁹(Zhang et al. 2019). Further work demonstrated that nitrate can increase the transcription and mRNA accumulation of AtNRT1.1 in the lateral root primordium, but inhibits the accumulation of AtNRT1.1 protein in the lateral root primordium, with a concomitant increase of auxin accumulation to enhance lateral root elongation, indicating that nitrate can modulate AtNRT1.1 at the post-transcriptional level in a tissue-specific manner⁹(Bouguyon et al., 2016). Recent work has uncovered that AtNRT1.1 displays auxin biosynthesis activity as well as auxin transport activity to modulate root branching when nitrate is available. AtNRT1.1 negatively regulates the expression of TAR2(one auxin biosynthetic gene) and LAX3 (auxin influx carrier), which inhibits auxin biosynthesis and represses the lateral root primordia development. However, this phenomenon is suppressed at high nitrate availability⁶(Maghiaoui 2020). These results suggest a vital role of AtNRT1.1 in regulating root system architecture in response to nutrient availability. Future efforts are needed to investigate the integrated roles of NRT1.1 providing an comprehensive overview of the functions of NRT1.1 in plants.

Calcium signaling triggered by NO₃ ^– at the transcriptional and post-transcriptional levels is a recent research avenue, as the components of calcium- and NO₃^–-signaling pathways (such as receptors, kinases, and transcription factors) were not identified until the current decade. Further transporters and signaling components still await discovery and their roles in functional and regulatory mechanisms remain to be elucidated. Ultimately, a greater understanding of the landscape of NO₃^–-sensing and -signaling pathways and how calcium-signaling networks connect with target biological processes is key to devising new biotechnological strategies to increase nitrogen-use efficiency in crops for sustainable agriculture.

Abbreviations

Table

ABA abscisic acid

ANR1 arabidopsis nitrate regulated 1

Ca^2+ calcium

CBL calcineurin B-like protein

CIPKs calcineurin B-like protein -interacting protein kinases CPKs, calcium-dependent protein kinases

CNGC15-NRT1.1 cyclic nucleotide-gated channel protein 15 interacting with nitrate transceptor

IP3 inositol 1, 4, 5-triphosphate

LR lateral root

NO3^– nitrate

NPC nuclear pore complex

PLC phospholipase C

PNR primary nitrate response

PP2C protein phosphatase 2C

Acknowledgments

We thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript.

Disclosure Statement

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR201807090168), the National Natural Science Foundation of China (Grant No. 31801930), Key Research and Development Project of Shandong Province (2019GNC106077) and the Major Science and Technology Innovation Project of Shandong province (Grant No.2018CXGC0208).