(Qi et al., 2018), and the excess ROS could be scavenged through enzymatic and non-enzymatic antioxidant defense systems (You and Chan, 2015). Accumulating investigations have revealed that ascorbic acid (AsA) is an essential compound of non-enzymatic antioxidant in plants, functioning in plant growth, hormone signaling, and stress response (Bulley and Laing, 2016; Mellidou and Kanellis, 2017; Vidal-Meireles et al., 2017). AsA plays especially critical roles in the fine control of ROS homeostasis to improve salt tolerance (Smirnoff, 2000; Shalata and Neumann, 2001; Zhang et al., 2012; Wang J. et al., 2013), implying that AsA has an essential modulation in salt response. Considering there are some reviews about ethylene-modulated salt response (Zhang M. et al., 2016), this mini review will focus on the advances in understanding the modulation of ethylene on AsA biosynthesis and ROS-scavenging under salinity stress.

# The Regulation of Ethylene on ROS Homeostasis Is Tightly Associated With the Plant Response to Salt Stress

Gaseous phytohormone ethylene plays an important role in mediating numerous specific growth and development processes (Wang et al., 2018), especially in response to various stress conditions (Wang F. et al., 2013; Dubois et al., 2018). The biosynthesis and signaling pathway of ethylene have been well established (Guo and Ecker, 2004). After recognition of ethylene by endoplasmic reticulum membrane-associated receptors, the interaction of ethylene receptors with CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) will be released, and the phosphorylation of CTR1 on ETHYLENE INSENSITIVE 2 (EIN2) will be liberated. Then, the C-terminal of EIN2 is generated by an unknown mechanism and is transported to cytoplasmic processing-body (P-body) to repress translation of EIN3 BINDING F-BOX1/2 (EBF1/2), which mediates the proteasomal degradation of EIN3 and EIN3-LIKE 1 (EIL1), resulting in the stability of EIN3/EIL1 proteins and promotion of ethylene response (Li et al., 2015). APETALA2/ETHYLENE RESPONSE FACTORS (AP2/ERFs) are one of the most important transcription factor families, regulating multiple developmental and stress response processes (Phukan et al., 2017), most of which are downstream targets of ethylene signaling (Liu et al., 2016).

Ethylene has long been known for modulating salt stress response (Cao et al., 2007). For instance, blocked ethylene signaling confers reduced salt tolerance to Arabidopsis (Achard et al., 2006; Peng et al., 2014). ein3 eil1 double mutants and other ethylene signaling-related mutants showed enhanced sensitivity to salt stress. In contrast to this modulation, ethylene displays a negative role in rice (Yang et al., 2015). OsEIL1 and OsEIL2 RNAi transgenic plants displayed increased salt tolerance. The regulation of ethylene biosynthesis also plays different roles in salt tolerance between Arabidopsis and rice (Jiang et al., 2013; Li et al., 2014). Ethylene Overproducer 1 (ETO1) plays a positive role in salt response through promoting ROS generation, followed with Na+/K+ homeostasis modulation in Arabidopsis. However, SALT INTOLERANCE 1 (SIT1) negatively regulates salt response due to activation on MITOGEN-ACTIVATED PROTEIN KINASE 3/6 (MPK3/6) in rice, which promotes ethylene and ROS overproduction (**Table 1**). Thus, the different mechanisms of ethylene-directed salt response between monocot and dicot plants remain in need of research. Subsequent advances indicate that ROS homeostasis is essential for ethylene regulation of plant growth and stress response (Steffens, 2014; Zhong et al., 2014; Yang et al., 2017). ROS is a double-edged sword during salt stress response. On the one hand, ROS act as important signal molecules to activate downstream metabolic pathways. Previous studies demonstrate that ROS burst via RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) and RbohF is essential for the Na+/K+ homeostasis in Arabidopsis (Ma et al., 2012), and ethylene-induced ROS production through transcriptional regulation on AtRbohF confers enhanced salt tolerance to the ethylene overproduced mutant eto1 (Jiang et al., 2013). On the other hand, ethylene signaling component EIN3/EIL1 activates ROS-scavenging gene expression to deter excess ROS accumulation and to increase salt tolerance (Peng et al., 2014). Similarly, the effects of ethylene signaling downstream factors on ROS are inconsistent during different stages of various stresses. For example, ERF74 promotes ROS burst in the early stages of various stresses through the regulation of gene expression of RbohD, followed with induction of ROS-scavenging-related genes (Yao et al., 2017). However, ethylene inducible factor TERF1 improves stress tolerance through reduced ROS content (Zhang H. et al., 2016). Therefore, fine-tuning of ethylene biosynthesis and signaling on ROS homeostasis are critical for salt tolerance.

Additionally, our previous studies verified several downstream regulators of ethylene signaling in salt response and ROS homeostasis. For example, ETHYLENE AND SALT INDUCIBLE ERF GENE 1 (ESE1), a direct target gene of EIN3, positively regulates salt tolerance and coordinates with EIN3 to activate downstream salt-related gene expression in Arabidopsis (Zhang et al., 2011); and JERF3, an ethylene-induced gene, enhances salt tolerance via direct modulation on the gene expressions of SUPEROXIDE DISMUTASE (SOD) and CARBONIC ANHYDRASE (CA) in tomato to eliminate ROS, which also confers drought and osmotic stress tolerance to transgenic rice with heterologous expression of JERF3 (Wu et al., 2008; Zhang et al., 2010; **Table 1**). Thus, identification of more ethylene signaling downstream regulators participating in ROS homeostasis under salt stress is necessary for elucidating the regulation of ethylene on ROS and salt response.

#### The Scavenging Role of AsA on ROS Homeostasis Contributes to Salt Tolerance

AsA, also known as vitamin C, is a low molecular weight antioxidant, functioning as a component of non-enzymatic scavenging of ROS in plant growth and stress tolerance (Smirnoff, 2000; Conklin, 2004; Akram et al., 2017). It has been reported that AsA improves salt tolerance in various species, including rice, potato, tomato, and citrus (Shalata and Neumann, 2001; Hemavathi et al., 2010; Kostopoulou et al., 2015; Qin et al., 2016). The L-galactose pathway is the main pathway of AsA biosynthesis in plants, and most of the genes in this pathway have been identified (Bulley and Laing, 2016). Investigations

TABLE 1 | Genes involved in ethylene- and AsA-mediated ROS homeostasis in response to salt stress.

also have elucidated the regulation via the L-galactose pathway of AsA biosynthesis, including the modulations on the AsA biosynthesis enzyme activities and stabilities at transcriptional and translational levels (Laing et al., 2015). One of these regulators is calmodulins-like 10, which interacts with AsA biosynthesis enzyme phosphomannomutase (PMM) to modulate enzyme activities and AsA pool (Cho et al., 2016), which suggested the role of calcium (Ca2+) in AsA biosynthesis. It has been known that Ca2+ signaling is triggered by ROS accumulation (Rentel and Knight, 2004) and Ca2+ wave is induced under salt stress (Choi et al., 2014; Liu et al., 2018). A chloroplast protein, QUASIMODO1 (QUA1), functions upstream of a thylakoid-localized Ca2+ sensor, CAS, to mediate Ca2+ signaling under salt stress (Zheng et al., 2017). Additionally, AsA could trigger increase of cytosolic Ca2+ in Arabidopsis as a signaling molecule (Makavitskaya et al., 2018), suggesting the association between Ca2+ sensor and AsA-mediated ROS scavenging during salt responses, and feedback regulation of Ca2+ signaling and ROS homeostasis.

The regulation factors of AsA biosynthesis also play a role in salt response. Our previous investigations have found that ethylene-induced factor AtERF98 enhances salt tolerance due to transcriptional activation on gene expressions of AsA biosynthesis enzymes, especially direct binding to the promoter of a key enzyme of AsA biosynthesis encoding gene VTC1 (Zhang et al., 2012). Meanwhile, we also identified the posttranscriptional modulation of COP9 SIGNALOSOME SUBUNIT 5B (CSN5B) on VTC1 in Arabidopsis (Wang J. et al., 2013; Li et al., 2016), elucidating a mechanism of light/dark effects on AsA contents. Loss-of-function mutant csn5b, with more AsA content and less ROS pool, displays increased salt tolerance, suggesting the positive regulation of AsA on salt response. Recent studies showed that salt induced zinc-finger protein SIZF3, which interferes with the interaction between CSN5B and VTC1, simultaneously promotes AsA accumulation and enhances salt tolerance (Li et al., 2018; **Table 1**). Thus, increasing AsA content is a potential approach for improving plant salt tolerance.

#### The Integration of Ethylene in AsA Production Finetunes ROS Homeostasis Under Salt Stress

As discussed above, both ethylene and AsA could enhance salt tolerance via regulation of ROS homeostasis. Previous reports have indicated that ethylene in many cases maintains a low level of ROS contents under salt stress through the enzymatic pathway (Wu et al., 2008; Peng et al., 2014; Zhang W. et al., 2016). Moreover, the non-enzymatic pathway of scavenging ROS also participates in ethylene-mediated salt response, such as AtERF98, suggesting that the modulation of ethylene on ROS elimination is alternatively dependent on non-enzymatic antioxidant (Zhang et al., 2012).

There are many environmental factors affecting AsA biosynthesis, such as light (Fukunaga et al., 2010), circadian rhythm (Dowdle et al., 2007), and high temperature (Richardson, 2004). CSN5B, identified in our previous studies (Wang J. et al., 2013), is a subunit of photomorphogenic COP9 signalosome (Gusmaroli et al., 2004), which acts together with COP1, COP10, and DET1 to repress photomorphogenesis (Yanagawa et al., 2004). This research suggest that CSN5B-regulated AsA biosynthesis is a part of photomorphogenesis. Intriguingly, ethylene has functions in COP1 nucleocytoplasmic partitioning (Yu et al., 2013, 2016), indicating a possible link between ethylene and light-regulated AsA biosynthesis. It was reported that ABA-INSENSITIVE 4 (ABI4) mediates AsA-regulated plant growth (Kerchev et al., 2011) and ethylene production via transcriptional

protein levels, activating gene expression of EIN3 direct binding targets (ESE1, SIED1, and POD) and ethylene response factors (JERF3 and ERF98) to regulate salt tolerance via ROS scavenging. ERF98 positively regulates salt tolerance via transcriptional activation of AsA biosynthesis gene VTC1. Moreover, CSN5B, a subunit of photomorphogenic COP9 signalosome, contributes to AsA biosynthesis and salt responses due to modulation on VTC1 degradation. SIZF3 also confers salt tolerance through mediating the interaction between CSN5B and VTC1. This research indicates that ROS accumulation under salt stress could be eliminated through enzymatic and non-enzymatic pathways, in both of which ethylene signaling is involved. However, the understanding of ethylene roles in AsA biosynthesis is yet limited. ABI4 negatively regulates ethylene synthesis and AsA production, which supply a possible mechanism coordinating ABA and ethylene to regulate AsA biosynthesis under salt stress. Ca2+ signaling could be induced by both ROS signaling and participates in AsA biosynthesis modulation through PMM. Arrows and lines with bars indicate activation and inhibition, respectively. Dotted lines indicate indirect regulations.

repression of ACS in Arabidopsis (Dong et al., 2016). In this regard, ethylene seems to have crosstalk with ABA to modulate AsA production (**Figure 1**). However, the mechanisms for these modulations are yet to be elucidated.

# CONCLUSION AND PERSPECTIVES

Emerging evidence provides the understanding of the roles of ethylene and AsA in salt tolerance through fine-tuning ROS homeostasis. Ethylene biosynthesis could be induced under salt stress, followed with ROS accumulation through transcriptional activation of Rbohs gene expression, in which ROS functions as a signal to regulate Na+/K+ homeostasis. Excessive ROS is toxic to plants, and ethylene also performs a scavenging role on ROS homeostasis under salt stress through signaling pathways, including the stability of EBF1/EBF2 and transcriptional regulation of EIN3/EIL1 on downstream direct or indirect regulators such as ESE1, SIED1, POD, JERF3, and ERF98. Ca2+ signaling, as a second messenger, could be induced by both ROS and AsA-mediated ROS balance, and participates in AsA biosynthesis modulation (**Figure 1**). The antagonistic effect of ethylene on ROS synthesis and scavenging under salinity stress is due to different functions of ROS at different developmental stages and in different tissues (Jiang et al., 2013; Peng et al., 2014). Additionally, non-enzymatic antioxidant AsA and the modulators involved in AsA biosynthesis confer to salt tolerance through reduced ROS accumulation (**Figure 1**; Wang J. et al., 2013; Qin et al., 2016; Li et al., 2018). However, the individual or crosstalk of ethylene and AsA regulation mechanisms on salt responses remain in need of further research. For example, although the components of the ethylene signaling pathway are conserved in Arabidopsis and rice (Yin et al., 2017), the underlying mechanisms of ethylene signaling in response to salt stress are different. Similarly, the effect of ethylene on plant growth is opposite in light and dark, such as hypocotyl elongation (Yu and Huang, 2017). Moreover, emerging research demonstrates that light plays a pivotal role in AsA synthesis

(Fukunaga et al., 2010; Wang J. et al., 2013). These findings suggest a complex network regulated by ethylene signaling under different growth conditions. Further engagement is needed to determine whether ethylene and light coordinate AsA production to maintain ROS homeostasis during salt response. Furthermore, it is widely recognized that ABA and ethylene are simultaneously involved in stress responses (Kumar et al., 2016). ABA signaling component ABI4 mediates AsAregulated plant growth (Kerchev et al., 2011) and inhibits ethylene biosynthesis (Dong et al., 2016; **Figure 1**). Nevertheless, the crosstalk between ethylene and ABA in the control AsA pool is unclear. Proper redox homeostasis is necessary for plant growth under salt stress; thus, making clear the detailed mechanisms of ethylene and AsA in maintaining ROS homeostasis will provide new insights for salt-tolerant genetic improvement.

#### REFERENCES

### AUTHOR CONTRIBUTIONS

RH proposed the concept. JW organized and drafted the manuscript. RH contributed to the editing of the manuscript. Both authors read and approved the manuscript.

# FUNDING

This work was supported by the National Key Research and Development Program of China (2016YFD0100604), the National Natural Science Foundation of China (31470366), Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences, and the Fundamental Research Funds for Central Non-profit Scientific Institution<PHONE_NUMBER>003).

ripening-associated ERF genes and their link to key regulators of fruit ripening in tomato. Plant Physiol. 170, 1732–1744. doi: 10.1104/pp.15.01859

negatively affect salt tolerance in rice. Plant Physiol. 169, 148–165. doi: 10.1104/ pp.15.00353

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wang and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Salinity and ABA Seed Responses in Pepper: Expression and Interaction of ABA Core Signaling Components

Alessandra Ruggiero1,2, Simone Landi1 , Paola Punzo1 , Marco Possenti3 , Michael J. Van Oosten2 , Antonello Costa1 , Giorgio Morelli3 , Albino Maggio2 , Stefania Grillo1 and Giorgia Batelli1 \*

#### Edited by:

Abel Rosado, University of British Columbia, Canada

#### Reviewed by:

Jorge Lozano-Juste, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Spain Simone Diego Castellarin, University of British Columbia, Canada

#### \*Correspondence:

Giorgia Batelli<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science

Received: 31 October 2018 Accepted: 25 February 2019 Published: 19 March 2019

#### Citation:

Ruggiero A, Landi S, Punzo P, Possenti M, Van Oosten MJ, Costa A, Morelli G, Maggio A, Grillo S and Batelli G (2019) Salinity and ABA Seed Responses in Pepper: Expression and Interaction of ABA Core Signaling Components. Front. Plant Sci. 10:304. doi: 10.3389/fpls.2019.00304 1 National Research Council of Italy, Institute of Biosciences and Bioresources (CNR-IBBR), Reaserch Division Portici, Portici, Italy, 2 Department of Agriculture, University of Naples "Federico II", Portici, Italy, 3 Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics (CREA-GB), Rome, Italy

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, including adaptation to stresses, fruit development and ripening. In seeds, ABA participates through its core signaling components in dormancy instauration, longevity determination, and inhibition of germination in unfavorable environmental conditions such as high soil salinity. Here, we show that seed germination in pepper was delayed but only marginally reduced by ABA or NaCl with respect to control treatments. Through a similarity search, pepper orthologs of ABA core signaling components PYL (PYRABACTIN RESISTANCE1-LIKE), PP2C (PROTEIN PHOSPHATASE2C), and SnRK2 (SUCROSE NONFERMENTING1 (SNF1)-RELATED PROTEIN KINASE2) genes were identified. Gene expression analyses of selected members showed a low abundance of PYL and SnRK2 transcripts in dry seeds compared to other tissues, and an upregulation at high concentrations of ABA and/or NaCl for both positive and negative regulators of ABA signaling. As expected, in hydroponically-grown seedlings exposed to NaCl, only PP2C encoding genes were up-regulated. Yeast two hybrid assays performed among putative pepper core components and with Arabidopsis thaliana orthologs confirmed the ability of the identified proteins to function in ABA signaling cascade, with the exception of a CaABI isoform cloned from seeds. BiFC assay in planta confirmed some of the interactions obtained in yeast. Altogether, our results indicate that a low expression of perception and signaling components in pepper seeds might contribute to explain the observed high percentages of seed germination in the presence of ABA. These results might have direct implications on the improvement of seed longevity and vigor, a bottleneck in pepper breeding.

Keywords: Capsicum annuum L., abscisic acid, PYR/PYL/RCARs, PP2Cs, SnRK2s, seed germination, seed viability

# INTRODUCTION

fpls-10-00304 March 15, 2019 Time: 17:20 # 2

Pepper (Capsicum spp.) is an economically important genus of the Solanaceae family, with a global production of 38.4 million tons including green fruits and dried pods, harvested from 3.7 million hectares in 20161 . In sweet pepper (Capsicum annuum L.), seed vivipary and short longevity are two major challenges of commercial production (Marrush et al., 1998; Lanteri et al., 2000; De, 2004; Sano et al., 2016).

Abscisic acid (ABA) plays a major role in the adaptation to stresses during the vegetative phase, as well as in the establishment of seed dormancy, seed longevity, and inhibition of germination under unfavorable conditions (Zhu, 2002; Finkelstein, 2013; Ruggiero et al., 2017). Seed germination starts with the uptake of water by imbibition of the dry seed, followed by embryo expansion. The uptake of water is triphasic with a rapid initial uptake (phase I, i.e., imbibition) followed by a plateau phase (phase II). A further increase in water uptake (phase III) happens only when germination occurs, as the embryo axis elongates and breaks through the covering layers, typically the endosperm and the testa (Manz et al., 2005). While in Arabidopsis thaliana testa and endosperm rupture occur simultaneously, in many Solanaceae seeds these two events are temporally distinct (Petruzzelli et al., 2003). Mechanical resistance from testa and endosperm dormancy appears to be the cause of nondeep physiological dormancy in seed model systems such as A. thaliana and Solanaceae species (Leubner-Metzger, 2003). Enzymes that facilitate testa rupture can be released by the endosperm and/or the radicle. β-1,3-glucanases (βGlu) facilitate endosperm rupture by breaking intercellular adhesion and causing cell separation. In pepper, the accumulation of βGlu occurs prior to radicle emergence. Unfavorable osmotic potentials, darkness and ABA inhibit endosperm rupture and βGlu accumulation in the micropylar cap of the seeds, the site of radicle emergence (Petruzzelli et al., 2003).

In non-dormant seeds, exogenous ABA also inhibits the transition from water uptake phase II to III and late embryo cell expansion, but does not affect phase I and II and testa rupture (Müller et al., 2006).

In Arabidopsis, ABA inhibition of germination is mediated by members of the core ABA signaling pathway as well as by ABFtype transcription factors.

Components of this signaling pathway have been isolated in the past decades, and include the PYRABACTIN RESISTANCE (PYR)/PYL (PYR1-LIKE)/REGULATORY COMPONENT OF ABA RESPONSE (RCAR), (hereafter referred to as PYLs), PROTEIN PHOSPHATASE2C (PP2C), and SNF1- RELATED PROTEIN KINASE2 (SnRK2) (Ma et al., 2009; Park et al., 2009). In the absence of ABA, clade A PP2Cs, a family of major negative regulators of ABA responses, bind to and inhibit SnRK2 kinases. The association keeps the kinases inactive by blocking their catalytic cleft and by dephosphorylating the activation loop (Soon et al., 2012). In response to environmental or developmental cues, ABA is perceived by PYL receptors and induces closure of two highly conserved β-loops that function as a gate and latch, and lock the pocket, creating a binding surface for the PP2Cs (Cutler et al., 2010; Moreno-Alvero et al., 2017). A conserved tryptophan residue in the PP2C inserts directly between the gate and latch, and functions to further lock the receptor in a closed conformation (Melcher et al., 2010; Santiago et al., 2012; Moreno-Alvero et al., 2017). SnRK2 kinases are thus released from PP2C inhibition, and can then phosphorylate many downstream effectors (Fujii et al., 2009), including the basic leucine-zipper transcription factors such as ABI5 (ABSCISIC ACID INSENSITIVE 5). A double snrk2.2/2.3 mutant displayed highly ABA-insensitive germination and increased seed dormancy (Fujii et al., 2007). Seed germination was resistant to 50-100 µM ABA in a sextuple pyr1/pyl1/2/4/5/8 mutant (Gonzalez-Guzman et al., 2012). Imbibed abi5 mutant seeds were able to perform the first stages of seed germination, but were arrested before the radicle penetration of the inner testa and endosperm (Lopez-Molina et al., 2002).

Genomic tools are now available to analyze the molecular details of these phenomena in pepper (Kim et al., 2014; Qin et al., 2014; Kim et al., 2017; Hulse-Kemp et al., 2018). The elucidation of the core ABA signaling pathway in model systems and the identification of key secondary regulatory proteins allows this knowledge to be applied to crop species (Klingler et al., 2010; Hou et al., 2016). Previous work has already characterized core components in Solanum lycopersicum (Gonzalez-Guzman et al., 2014; Chen et al., 2016) and a pair of pepper PP2C/PYL proteins has been previously functionally analyzed (Lim and Lee, 2016). Different studies have shown the applications of ABA signaling core components to enhance plant stress tolerance, through either genetic engineering or chemical approaches in crops (Okamoto et al., 2013; Rodriguez and Lozano-Juste, 2015; Zhang et al., 2016; Vaidya et al., 2017). Pepper presents a potential practical application of these methods for genetic improvement that can directly benefit breeders, producers, and consumers.

Here, we report on ABA and NaCl sensitivity in pepper seeds and plants as well as the identification of components of the C. annuum PYL, PP2C, and SnRK2 gene families. We have characterized pepper's sensitivity to ABA and NaCl at the germination stage by evaluating gene expression and the potential for in vivo interaction among the ABA signaling components. We performed yeast two hybrid assays among putative pepper core components and with Arabidopsis orthologs to confirm the ability of these proteins to function in the ABA signaling cascade. We also verified interactions in protoplasts using a bi-molecular florescence assay.

#### MATERIALS AND METHODS

#### Plant Material

Capsicum annuum L. Quadrato D'Asti giallo (2480) seeds were provided by "S.A.I.S. Spa" (Italy), while seeds of the genotypes Corno di Toro rosso (QSB294TS), (refer to as

1www.fao.org

Corno rosso), Corno di Toro giallo (QSB296TS) (refer to as Corno giallo), Friariello (Nocera selection, PGM283), Nocera rosso (Japanese selection, QPA1909TS), Nocera giallo (Japanese selection, QPA1310TS), and Marconi rosso (R15020) were kindly gifted by "La Semiorto Sementi s.r.l." (Italy).

For germination analyses, seeds were sown on solid MS medium (1X MS Salts including vitamins, 15 g/L Plant agar, pH 5.7) in the presence of ABA (1, 5, and 10 µM) or NaCl (25, 50, and 100 mM). Germination was scored daily in terms of radical emergence and fully expanded cotyledons.

For seedling sensitivity to ABA and NaCl, seedlings were transferred from germination media after 9 days and were grown on vertical plates on solid MS medium (1X MS Salts including vitamins, 5 g/L Sucrose, 15 g/L Plant agar, pH 5.7) in the presence of 100 mM NaCl or 20 µM ABA. Root length was scored every 2 days, shoot weight and photographs were taken after 5 days of growth.

In hydroponic culture, pepper seedlings were grown in solution containing: 1.5 mM Mg(NO3)2·6H2O, 3.4 mM Ca(NO3)2·4H2O, 1 mM KNO3, 1.8 mM K2SO4, 1.5 mM KH2PO4, and 14 mg/L Hidromix (Valagro, Italy), pH 6.2. The solution was changed weekly. After 17 days of culture, NaCl 200 mM was added to the solution to impose salt stress. After 3 h of salt treatment, shoots and roots of each condition were collected separately.

#### RNA Isolation, cDNA Synthesis, and qRT-PCR

Total RNA was extracted from seeds, shoots and roots (100 mg) using RNeasy R Plant Mini kit (Qiagen, Germany) following the manufacturer's instructions. RNA quantity was measured spectrophotometrically by NanoDrop ND-1000 Spectrophotometer (NanoDropTechnologies, United States), and integrity was verified on a denaturing agarose gel. One microgram of total RNA was DNase-treated and reverse transcribed using QuantiTect R Reverse Transcription kit (Qiagen, Germany) according to manufacturer's instructions. For gene expression analyses, the complementary DNA was diluted 1:20 and 2 µL of diluted cDNA were used for each qRT-PCR reaction, performed with 6.25 µL of 1X Platinum R SYBR R Green qPCR SuperMix (Thermo Fisher Scientific, United States) and 1.75 µL of primer mix (4.28 µM) in a 12.5 µL PCR reaction. Primers used are listed in **Supplementary Table S1**. Reactions were performed with ABI 7900 HT (Applied Biosystems, United States). Cycling conditions were: 10 min at 95◦C, followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. Three or four biological replicates per treatment, each with three technical replicates were tested. PCR product melting curves were analyzed to confirm the presence of a single peak, indicative of one PCR product per primer couple assayed. For relative quantification of gene expression, Capsicum annuum eukaryotic initiation factor 5A2 (EIF5A2) (Acc.no. AY484392) was used as endogenous reference since its expression was found stable in all the analyzed tissues and treatments as also reported by Wan et al. (2011). Quantification of gene expression was carried out using the 2 −11Ct method (Livak and Schmittgen, 2001) and reported as relative expression levels, compared to control conditions as internal calibrator.

For absolute qRT-PCR total RNA was extracted from dry seeds, germinating seeds (i.e., seeds incubated for 4 days on control medium), shoots and roots from seedlings grown in hydroponic system in control condition. RNA isolation, cDNA Synthesis and qRT-PCR were performed as already mentioned above. All primer pairs (reported in **Supplementary Table S1**) were tested by PCR. A single product of the correct size for each gene was confirmed by agarose gel electrophoresis. The amplified fragment of each gene was subcloned into the pGEM R -T Easy vector (Promega, United States) and used to generate standard curves by serial dilutions. Results were analyzed using the ABI PRISM 7900HT Sequence Detection System, Version 2.3. Analysis of variance (ANOVA) on absolute qRT-PCR data was carried out using the SPSS software package (SPSS 19 for Windows, SPSS Inc., an IBM Company, United States). When ANOVA indicated that a single factor or their interaction was significant, mean separation was performed using the Duncan's multiple range test at p < 0.05 on each of the significant variables measured.

#### Yeast Two-Hybrid Assay

For yeast two-hybrid experiments, the prey plasmid pGADT7 and the bait plasmid pGBKT7 (Clontech, United States) were used. The full-length coding sequence of CaABI and CaHAI were PCR amplified using RNA prepared from seeds and cloned in frame into pGADT7 between EcoRI and XhoI, SmaI and XhoI restriction sites, respectively. The full-length coding sequence of CaPYL2 and CaPYL4 were PCR amplified using RNA prepared from leaves and cloned in frame with the GAL4 binding domain (BD) of pGBKT7 digested with SmaI and PstI. The full-length coding sequence of CaPYL8, CaSnRK2.3, and CaSnRK2.6 were PCR amplified using RNA prepared from seeds and cloned in frame into pGBKT7 between EcoRI and BamHI, EcoRI and SalI, EcoRI, and BamHI restriction sites, respectively. Plasmids were sequenced to rule out PCR-induced mutations. Primers used for PCR amplification of the mentioned genes are listed in **Supplementary Table S2**. The bait and prey plasmids containing Arabidopsis thaliana genes were previously described (Park et al., 2009; Hou et al., 2016).

The bait and prey plasmids were transformed into yeast strain AH109 (Clontech, United States) using the Lithium acetate/Polyethylene glycol method (Bai and Elledge, 1997). The self-activation test was performed prior to the testing of combinations of interest as reported in Docimo et al. (2016). After verifying that the bait and prey plasmids were not showing self-activation, co-transformations to verify interactions were performed. Transformed colonies containing bait and prey plasmids were selected on synthetic drop-out medium lacking tryptophan and leucine (-W/-L). Co-transformants were grown overnight in liquid culture lacking tryptophan and leucine (-W/- L). For the interaction between bait and prey, an equal amount of cells was spotted on medium lacking tryptophan, leucine, histidine and adenine (-W/-L/-H/-A). Positive and negative controls were also performed as indicated in the figure legend.

# Bimolecular Fluorescence Complementation Assay

fpls-10-00304 March 15, 2019 Time: 17:20 # 4

The CDS of CaPYL2 and CaPYL4 were fused downstream of N-terminal region of YFP, while CaHAI was fused downstream of the C-terminal region of YFP, using pUGW0 vectors (Nakagawa et al., 2007). Leaf protoplasts were prepared and transformed according to Pedrazzini et al. (1997), using 3 weeks old N. tabacum plants. DNA (40 µg of each construct) was introduced into 1 × 106 protoplasts by PEG-mediated transfection. After 16 h incubation in the dark at 25◦C, each interaction was split in two and one was treated with 50 µM of ABA. After the incubation time, YFP fluorescence in protoplast cells was detected by confocal microscopy.

# Confocal Imaging

Confocal microscopy analyses were performed on an Inverted Z.1 microscope (Zeiss, Germany) equipped with a Zeiss LSM 700 spectral confocal laser-scanning unit (Zeiss, Germany). Samples were excited with a 488 nm, 10 mW solid laser with emission split at 505 nm for YFP.

# Bioinformatics

Sequences of pepper (Capsicum annuum) ABA receptors (PYL/PYR/RCAR), PP2Cs and SnRK2s were found using a multiple database search to identify potential members of these families. Tomato (Solanum lycopersicum) and Arabidopsis thaliana sequences were previously identified by other authors (Schweighofer et al., 2004; Gonzalez-Guzman et al., 2014) and obtained at https://solgenomics.net and https://www.arabidopsis. org, respectively. Pepper sequences were obtained using a BLAST P approach at http://pepperhub.hzau.edu.cn/pegnm/ database, using each A. thaliana and tomato sequence as queries. The obtained hits were filtered using a blast score cut-off ≥ 150. Alignments and phylogenetic analyses were performed using the software MEGA version 6 (Tamura et al., 2013). Sequence alignment was achieved using the MUSCLE algorithm, using a maximum number of interactions equal to 32. Phylogenetic trees were constructed using the maximum likelihood method with the substitution JTT model gamma distributed. The test of phylogeny was performed using the bootstrap method with a number of replications equal to 100.

For cloned PP2Cs, alignments of multiple amino acid sequences were carried out using Clustal W2 . The alignment results were marked using BOXSHADE 3.21 software3 .

# RESULTS

#### Pepper Seed Germination in Presence of ABA and NaCl

To verify the sensitivity of Capsicum annuum to abiotic stress and ABA treatments at the seed stage, we scored the germination percentage of commercial seeds of Quadrato D'Asti giallo (QA),

2https://www.genome.jp/tools-bin/clustalw

3http://www.ch.embnet.org/software/BOX\_form.html

a high yielding variety among the most commonly cultivated for marketing in Italy and abroad. The germination rates in terms of radicle emergence and cotyledon expansion of seeds in presence of ABA or NaCl were analyzed in detail (**Figure 1**). After 15 days, seeds treated with 1 or 5 µM ABA reached virtually 100% germination, while those treated with 10 µM ABA showed 62% seed germination (**Figure 1A**). The seeds incubated with 10 µM ABA had complete inhibition of cotyledon expansion, while the presence of 5 µM ABA resulted in a 6 day delay compared to controls or 1 µM ABA (**Figure 1B**). The radicle emergence was not affected by salt treatments except for 100 mM NaCl, which caused a 1 day delay of germination compared to no or lower NaCl concentrations (**Figure 1C**). Sodium chloride also delayed cotyledon expansion. At 100 mM NaCl, only 63% of the seedlings had fully expanded cotyledons after 15 days of incubation, while at 0, 25, and 50 mM 90% of the cotyledons were fully expanded (**Figure 1D**). The delayed cotyledon expansion caused by the highest concentrations of ABA and NaCl was also visible early as 8 days (**Figure 1E**). Therefore, the presence of NaCl or ABA had little effect on QA seed germination at low and medium concentrations with a clear effect only at high concentrations. A similar behavior was observed in other tested varieties, which showed significant germination reduction at 10 µM ABA and 100 mM NaCl (**Supplementary Figure S1**).

We also tested response of seedlings to ABA and NaCl to verify sensitivity at different developmental stages. We therefore scored root growth of QA seedlings germinated on control media and subsequently transferred to plates containing NaCl or ABA for 5 days (**Figure 2**). As shown in **Figures 2A,B**, root growth was inhibited at 20 µM ABA. In particular, 2 days after transfer, the seedlings grown in presence of 20 µM ABA showed a 16% increase of the initial root length compared to a 33% increase in 100 mM NaCl and a 40% increase in plants grown in the control medium. After 5 days of incubation, there was a 30% difference in root growth between seedlings grown on ABA and no or NaCl treatments. Measurements of shoot weight at the end of the experiment allowed for a discrimination of the treatments, showing a reduction in plants treated with NaCl 100 mM or ABA 20 µM compared to controls (**Figure 2C**).

#### Capsicum annuum PYL, PP2C, and SnRK2 Identification

Using annotated tomato and Arabidopsis protein sequences, we identified putative orthologs in pepper genome (C. annuum L\_Zunla-1, Qin et al., 2014) of ABA signal transduction core components: PYR/PYL/RCAR (PYLs), clade A PP2Cs and SnRK2s (**Figure 3**). A high bootstrap was observed for the three analyzed families, with C. annuum homologs showing a closer similarity to the tomato counterparts. Ten PYL genes (**Figure 3A**) were identified and clustered in the classical three subfamilies (Ma et al., 2009), with the exception of Capana02g001761, which clustered with Solyc02g076770. A multiple aminoacid sequence alignment of Ca- and AtPYLs showed that functional residues in the conserved loops (CL1-CL4) were well conserved (**Supplementary Figure S2**).

Seven genes encoding putative clade A PP2Cs showed a partition in 2 subfamilies (**Figure 3B**); the first included Capana06g000398, Capana05g002193 and Capana03g002491, clustering with Arabidopsis AIP1, HAI3, HAI1, AHG3, and AHG1; Capana03g000145, previously characterized as CaADIP1 grouped with Arabidopsis ABI1, ABI2, HAB2, and HAB1, as previously shown (Lim and Lee, 2016) and with Capana07g000875, Capana12g000483, and Capana08g000504. We focus our studies on three CaPP2Cs representatives: Capana03g002491, Capana06g000398, and Capana07g000875 (circled in red, **Figure 3B**). A multiple alignment of Capana03g002491 deduced amino acid sequences with AtAHG1, Solyc03g006960, AtABI1 and AtHAB1 showed that most of the functional residues were well conserved, including those necessary for the Mn/Mg++ interaction and the interaction with PYLs (**Supplementary Figure S3**). Similarly to AtAHG1, Capana03g002491 lacks the tryptophan that in other clade A PP2Cs participates to the binding with ABA (W385 in AtHAB1, W300 in AtABI1), which in Arabidopsis appears to be a unique feature of AHG1. Thus, we named Capana03g002491 as CaAHG1 (**Figure 3B**).

To study protein interactions, Capana06g000398 and Capana07g000875 were isolated and cloned using RNA extracted from seeds. The proteins encoded by the cloned coding sequences showed only a few conservative amino acid replacements with similar properties (**Supplementary Figure S4**). A notable exception is the substitution of a phenylalanine involved in the Van der Waal contacts with PYLs (F306 in ABI1, Yin et al., 2009), which in sequences from Solanaceous plants is replaced by a serine (S), indicating possible modifications in the binding with regulatory PYLs. Furthermore, the cloned Capana07g000875, which corresponds to XM\_016724784.1, one of the four predicted splicing variants of this gene, has a shorter protein sequence compared to other PP2Cs aligned, therefore it lacks two conserved D residues required for the Mn/Mg++ ions interaction (**Supplementary Figure S4**). On the basis of protein homology and conserved functional residues we named Capana06g000398 as a putative CaHAI and Capana07g000875 as

CaABI (circled in red, **Figure 3B**). Nine genes encoding SnRK2s were clustered in the three classical sub-families (**Figure 3C**, Umezawa et al., 2010), and were all already annotated in the genome. Similarly to tomato, two genes encoding subfamily III SnRK2s appear to be present in the genome, compared to three genes in Arabidopsis. Alignments of the domain II in the C-terminal region of the SnRK2 proteins separate pepper SnRK2s in SnRK2a and SnRK2b subfamilies (**Supplementary Figure S5**).

# PYL, PP2C, and SnRK2 Gene Expression in Different Tissues

For gene expression analyses, we selected three PYLs, one for each subfamily (CaPYL2, CaPYL4, CaPYL8), three PP2Cs from different subclades (CaABI, CaAHG1, CaHAI), the two SnRK2s from subclass III (CaSnRK2.3, CaSnRK2.6) and one from subclass I (CaSnRK2.4) (**Figure 3**, red circles).

To verify organ specific expression, we performed absolute quantification through qRT-PCR in dry seeds, germinating seeds, shoots and roots (**Figure 4**). CaPYL2 showed low expression in seeds, both dry and germinating, and roots, with highest levels of expression detected in shoots (about 10,000 copies/µL cDNA). CaPYL4 had low expression in dry seeds (about 400 copies/µL cDNA), higher in germinating seeds and shoots, and the highest expression among the tested CaPYLs in roots (about 48,000 copies/µL cDNA) (**Figure 4A**). Regarding expression of PP2Cs in dry seeds, CaABI and CaHAI showed the lowest (about 470 copies/µL cDNA) and highest (about 10,000 copies/µL cDNA) amount, respectively (**Figure 4B**), while similar expression values in the other organs were detected. All three selected SnRK2 genes showed low expression in dry seeds. CaSnRK2.4 had low expression also in germinating seeds (around 400 copies/µL cDNA) and roots (around 800 copies/µL cDNA) while CaSnRK2.6 showed the highest steady state level (21,000 copies/µL cDNA) in shoots (**Figure 4C**).

# Regulation by NaCl and ABA of PYL, PP2C, and SnRK2 Expression

We studied the expression of selected genes in QA seeds incubated for 4 days with ABA (1, 5, 10 µM) or NaCl (25, 50, 100 mM). ABA or salt treatments resulted in up-regulation of positive ABA signaling components such as PYLs and SnRK2s (**Table 1**). In particular, CaPYL4 expression was significantly upregulated by the highest concentration of ABA, whereas CaPYL8 was upregulated by all NaCl treatments. CaPYL2 expression level was below detection. CaSnRK2.6 was up-regulated in all tested treatments except for the lowest ABA concentration, while

CaSnRK2.3 was induced by ABA 10 µM, NaCl 50 and 100 mM. CaSnRK2.4 was up-regulated by ABA 10 µM and all NaCl treatments. CaHAI was the only PP2C gene up-regulated by ABA and all NaCl treatments while CaAHG1 was up-regulated only by ABA 1 µM. We also tested shoot and root expression of QA seedlings grown in hydroponic solution and subjected to 3 h salt stress treatments (**Table 2**). In this case, the three tested PP2C genes were significantly up-regulated in both tissues. By contrast, positive regulators were less responsive; CaSnRK2.6 was the only SnRK2 up-regulated in shoot. Notably, CaPYL4 was strongly down-regulated after NaCl treatment in both shoots and roots.

#### Interaction Between PYLs, PP2Cs, and SnRK2s

The key function of ABA receptors upon ABA binding is their ability to interact with and inhibit PP2Cs, releasing SnRK2s from inhibition (Fujii et al., 2009; Park et al., 2009). We therefore tested whether selected CaPYLs, CaPP2Cs and CaSnRK2s were capable of interaction with each other or with Arabidopsis members of the ABA signaling cascade in the yeast two hybrid assay. CaPYLs and CaSnRK2s were fused to the GAL4 Binding Domain (BD) and the PP2Cs CaABI and CaHAI to the GAL4 activation domain (AD) and used to test multiple combinations (**Figure 5**). CaPYL4 interacted with AtABI1 and AtPP2CA irrespective of the presence of ABA, while CaPYL2 interaction with AtABI1, AtABI2 and AtPP2CA was only observed in presence of the hormone (**Figure 5A**). The ability of CaHAI to interact with AtPYLs in presence or not of ABA was also tested (**Figure 5B**). Yeast co-transformed with CaHAI and, respectively, AtPYL6, AtPYL7, AtPYL8, AtPYL9, AtPYL11, AtPYL12 and AtPYL13 was able to grow on selective media (–W/–L/–H/–A). Interactions between AtPYL6, AtPYL11 and CaHAI were weak without ABA, but increased markedly when ABA was

present. Furthermore on selective media containing ABA, the yeast co-transformed with AtPYL3/CaHAI, AtPYL4/CaHAI and AtPYL10/CaHAI combinations was able to grow in addition TABLE 2 | Relative quantification of gene expression measured by qRT-PCR of selected genes in roots and shoots of Quadrato D'Asti giallo seedlings grown in hydroponic system after 3 h of 200 mM NaCl treatment.

All data are expressed relative to control treatment as mean ± SD (n = 3). Asterisks represent significance levels using Student t-test; ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.005. B.D., below detection.

to other combinations mentioned above. The combination AtPYL5/CaHAI showed a weak growth on the selective media with ABA (**Figure 5B**). No interaction was observed for any transformation with the cloned CaABI and each of the AtPYLs (**Supplementary Figure S6**) or CaPYL2/4/8 (**Figure 5C**). In the test between CaPYLs and CaHAI, we observed interaction in presence of ABA with CaPYL2/4/8, while without ABA only the combination CaPYL8/CaHAI showed growth (**Figure 5C**). The interactions between PP2Cs and SnRK2s of C. annuum and Arabidopsis were also analyzed in yeast (**Figures 5D–F**). When we tested combinations between AtSnRK2s and CaPP2Cs, only the yeast co-transformed with the member of subfamily III AtSnRK2.6 and CaHAI was able to grow on the selective media (**Figure 5D**). Similarly, we observed interaction between CaSnRK2.3/2.6 and AtABI1 and AtPP2CA (**Figure 5E**). A positive interaction between CaPP2Cs and CaSnRK2s was observed when yeast was cotransformed with CaHAI and CaSnRK2.3 or CaSnRK2.6, while the cloned CaABI was incapable of binding to SnRK2s in yeast (**Figure 5F**). CaABI did not interact with any of the Arabidopsis or pepper ABA receptors and SnRK2s in yeast (**Figures 5C,D,F** and **Supplementary Figure S6**), although the

TABLE 1 | Relative quantification of gene expression measured by qRT-PCR of selected genes in seeds of Quadrato D'Asti giallo incubated for 4 days on control medium and media with different concentrations of ABA or NaCl.

All data are expressed relative to control treatment as mean ± SD (n = 4). Asterisks represent significance levels using Student t-test; ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.005.

FIGURE 5 | Interaction test of PP2Cs with PYLs and SnRK2s in the yeast two-hybrid assay. (A) AtPP2Cs fused to the GAL4 Activation Domain (AD) were co-transformed in yeast with CaPYLs cloned in frame with the GAL4 binding domain (BD) of pGBKT7, with combinations shown in figure. (B) CaHAI fused to the AD in pGADT7 was expressed in yeast with AtPYLs in pBDGAL4, the different combinations are shown. (C) CaPP2Cs were fused to the AD in pGADT7 and co-transformed with CaPYLs cloned in pGBKT7. (D) CaPP2Cs in pGADT7 were co-transformed in combination with AtSnRK2s cloned in pGBKT7. (E) AtPP2Cs in pACT2 were co-transformed in yeast with CaSnRK2s in pGBKT7, with combinations shown in figure. (F) Co-transformants of yeast containing CaPP2Cs fused to the AD in pGADT7 in combination with CaSnRK2s in pGBKT7. Co-transformations of the pepper PYL/SnRK2/PP2C orthologs with appropriate complementary empty vectors are shown as negative controls. Yeast cells grown on synthetic media (–W/–L) and on synthetic, selective media without (–W/–L/–H/–A) or, where indicated, with 50 µM ABA (–W/–L/–H/–A+ABA) are shown. Pictures were taken after 3 days of incubation at 30◦C.

protein was produced in yeast, as verified through westernblot by the presence of a signal of the expected molecular weight (**Supplementary Figure S7**). We confirmed in a plant system the interaction between CaHAI and CaPYL2/4 in presence of ABA. We used a split-reporter system in tobacco protoplasts by transient co-expression of CaHAI and CaPYL2/4 fused to complementary fragments of the YFP. A reconstituted fluorescence signal was observed for both interactions only in presence of ABA (50 µM, **Figure 6**), thus confirming the results obtained in yeast. The interaction CaHAI-CaPYL2 was observed within 5 min of incubation with ABA, while a 2 h incubation was allowed to detect the CaHAI-CaPYL4. All negative controls with or without ABA are reported in **Supplementary Figure S8**.

We also investigated the subcellular localization of the proteins CaABI, CaHAI, CaPYL2, and CaPYL4 (**Supplementary Figure S9**). CaABI, CaPYL2, and CaPYL4 showed a nuclear and cytoplasmic localization, while for YFP-CaHAI the fluorescence signal was present only in the nucleus (**Supplementary Figure S9**).

#### DISCUSSION

Seed germination and seedling growth are adversely affected by ABA and NaCl. Capsicum annuum is classified as a moderately salt-sensitive species (De Pascale et al., 2003; Aktas et al., 2006; Penella et al., 2016). Here, we have shown that pepper cv. Quadrato D'Asti giallo (QA) had reduced seed germination and a complete inhibition of cotyledon expansion at 10 µM ABA. Similarly, the presence of 100 mM NaCl caused a 1 day delay in seed germination and reduced cotyledon expansion. Therefore, QA seeds displayed an inhibited germination only at the highest NaCl or ABA concentrations applied (**Figure 1**). At seedling stage, we observed a reduction of root length and shoot weight in seedlings grown in the presence of 100 mM NaCl (**Figure 2**), consistent with previous reports of a reduced vegetative growth in presence of NaCl (Yildirim and Güvenç, 2006).

NaCl-induced inhibition of germination in pepper is mainly due to the osmotic component of salt stress (Chartzoulakis and Klapaki, 2000; Demir and Mavi, 2008), which is counteracted in plants by employing ABA-dependent mechanisms (Cutler et al., 2010; Nakashima and Yamaguchi-Shinozaki, 2013).

Differences in sensitivity to ABA/NaCl between developmental stages/tissues, which may also contribute to explain the known phenomenon of pepper vivipary and low seed longevity, might be at least partially due to the expression/activity of ABA signaling components. Therefore, through a sequence similarity search, we identified putative pepper orthologs of PYLs, clade A PP2Cs and SnRK2s. Reflecting a high degree of conservation of these families among land plants, cluster analysis showed the classical organization in 3 subfamilies for pepper PYLs and SnRK2s. However, similarly to the tomato PYLs, no close relative for the Arabidopsis subgroup AtPYL11/12/13 was found (Gonzalez-Guzman et al., 2014). Capana02g001761, annotated as CaPYL12 like, did not cluster in any of the classical three subfamilies (**Figure 3A**), and was in a clade with the putative tomato ortholog Solyc02g076770, also ungrouped in the study of Gonzalez-Guzman et al. (2014). Capana02g001761, as its tomato ortholog, lacked key aminoacids, including residues of conserved loop 2 (CL2), CL3, and CL4 (**Supplementary Figure S2**). In contrast, presence of these conserved residues, as well as the ABA-dependent interaction with PP2C observed in yeast and protoplasts suggests that CaPYL2 and CaPYL4 are functional ABA receptors.

Additionally, the functional residues or domains were well conserved in the deduced sequences of three selected CaPP2Cs (**Supplementary Figures S3**, **S4**). Similarly to Arabidopsis AHG1 and Solyc03g006960, CaAHG1 lacks the tryptophan (W385 in HAB1, W300 in ABI1) that in other clade A PP2Cs participates to the binding with ABA (Weiner et al., 2010).

The domain II in the C-terminal region of the SnRK2 proteins is critical for PP2C interaction and ABA responsiveness (Kobayashi et al., 2005; Yoshida et al., 2006). Seven pepper SnRK2s (Capana06g001594, Capana01g000732, Capana08g002441, Capana02g003135, Capana05g000287, Capana12g002655, Capana04g000996) belong to subgroup 3 and 2, which in Arabidopsis can be strongly or weakly activated by ABA; while Capana08g001898 and Capana00g002391 cluster with subgroup 1, containing SnRK2s that may not be activated by ABA treatment (**Figure 3C** and **Supplementary Figure S5**; Kulik et al., 2011).

Quantification of gene expression of selected genes in different organs showed differences among family members (**Figure 4**).

CaPYL2 was highly expressed in shoots, while CaPYL4 and CaPYL8 showed highest expression in roots. These data are consistent with previous studies in tomato (Gonzalez-Guzman et al., 2014) and soybean (Bai et al., 2013). Interestingly, differences in transcript levels of the tested PP2Cs were observed only in seeds. CaHAI had highest steady state expression levels in dry seeds. By contrast, CaABI had the lowest expression in dry or germinating seeds and similar expression in all vegetative tissues. Finally, SnRK2 kinases, including the two subclass III members, also had lowest expression in dry seeds. In other species, SnRK2s were shown to be highly expressed in seeds (Fujii et al., 2007; Nakashima et al., 2009; Lou et al., 2017). Considering the established role of these kinases in plant responses to abiotic stress (Zhu, 2016), the low SnRK2 expression in seed suggests an ineffective mechanism of ABA signaling that may affect sensitivity to ABA at the germination stage. However, it is possible that other SnRK2s genes, not tested here, might be highly expressed in seeds. In addition, activity of ABA signaling components is regulated post-translationally through ABA binding, protein interaction and/or phosphorylation/dephosphorylation. Thus, the gene expression in specific organs or in response to treatments only provides an initial indication of the effectiveness of this mechanism, which will have to be verified by measurements of protein activity.

In seeds, 10 µM ABA treatment induced up-regulation of PP2C CaHAI, indicating a possible negative feedback

ABA. Scale bars = 20 µm in all panels.

at high ABA concentrations, as well as of all the tested SnRK2s. ABA-induced up-regulation of SnRK2s has been reported in the case of vegetative tissues, and is thought to derive from a self-amplification mechanism of ABA signal transduction components (Wang et al., 2018). Interestingly, 10 µM ABA treatment also induced CaPYL4 expression, while in vegetative tissues PYLs are commonly down-regulated by ABA (Wang et al., 2018).

In seedlings, CaPYL4 was significantly down-regulated after NaCl treatment in shoots and roots, while CaPYL8 did not change significantly (**Table 2**). Similarly, in tomato seedlings exposure to dehydration caused repression of SlPYL4 expression, while SlPYL8 showed no change in gene expression (Sun et al., 2011). By contrast, all genes within the SlPP2C gene family were significantly up-regulated by dehydration (Sun et al., 2011). In rice, expression of 10 OsPP2C genes in plants treated with 150 mM NaCl was up-regulated (Xue et al., 2008). Consistently, we observed NaCl-induced up-regulation for CaABI, CaHAI, and CaAHG1 (**Table 2**).

Protein-protein interaction assays through Y2H between pepper core signaling components or with Arabidopsis orthologs showed ABA-dependent and -independent interactions (**Figure 5**), as observed in examples from other crops (Wang et al., 2018). CaHAI showed an interaction pattern similar to the Arabidopsis orthologs (Bhaskara et al., 2012). In BiFC, interaction between CaHAI and CaPYL2 or CaPYL4 were both ABA-dependent and localized to the nucleus and cytoplasm, indicating that both transcriptional and immediate responses to ABA can be regulated by these complexes (**Figure 6**).

Although all the data here gathered points toward CaPYL2/4, CaHAI, and SnRK2.3/2.6 as ABA receptors, PP2C phosphatases and SnRK kinases, respectively, in vitro activity assays are needed to truly probe this point.

Notably, a CaABI transcript, annotated as splicing isoform 4 (XM\_016724784.1), amplified from dried/germinating seeds, encoded a truncated protein lacking two conserved D residues required for the Mn/Mg++ ions interaction. When used for interaction assays was not capable to complex with PYLs or

SnRK2s, suggesting that this variant may not be functional. Alternative splicing of pre-mRNAs constitutes an additional layer of regulation of gene expression and a way to expand the proteome diversity or regulate protein functionality in specific developmental/environmental situations, or cell types (Laloum et al., 2018). In plants, intron retention is indicated as the most common alternative splicing type (Ner-Gaon et al., 2004). Stress and ABA-response regulatory genes are known to be most prone to alternative splicing phenomena, which may produce active vs. inactive protein variants (Laloum et al., 2018). Specifically, splicing variants have been described in the case of the Arabidopsis HAB1 PP2C (Ling et al., 2017). The ratio between the full-length, functional HAB1 protein and the truncated splice form resulting from intron retention are regulated by the ABA levels and are involved in the fine-tuning of the ABA signaling cascade (Wang et al., 2015). The splicing variant of CaABI expressed in seeds may result from a similar mechanism of balance of phosphatase activity, and may reflect a developmental rather than environmental regulation between protein isoforms.

#### CONCLUSION

In conclusion, pepper seeds retained high germination percentages in presence of NaCl and ABA. This may be due to low expression in seeds of ABA signaling components such as CaABI, CaPYL2, CaPYL4, CaSnRK2.3, CaSnRK2.6. The expression of tested PYLs and SnRK2s was comparatively higher in vegetative tissues, and this correlated with restored sensitivity to exogenous ABA and salinity. Expression of a splice variant of CaABI encoding a truncated protein unable to interact with other tested signaling components may also impair ABA signaling cascade in seeds. The alleles controlling the expression of these signaling components in maturing and dry seed are potential traits that can be manipulated for the purposes of improving pepper low seed storability through molecular breeding and gene editing.

#### REFERENCES

# DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the **Supplementary Files**.

#### AUTHOR CONTRIBUTIONS

AR, SG, and GB designed the work. AR, SL, PP, and MP performed the experiments and prepared the figures. AR, SL, MVO, and AC analyzed the data. GM and AM participated in the experimental design and data interpretation. AR, MVO, and GB wrote the manuscript, with inputs from all authors. All the authors have read and approved the manuscript.

### FUNDING

This work was supported by the Italian Ministry of University and Research, under Grant No. PON02\_00395\_3215002 (GenHORT).

#### ACKNOWLEDGMENTS

We wish to thank Dr. V. Cirillo (University of Naples, Federico II) for help with the statistical analyses and Mr. G. Guarino (CNR-IBBR) for help with figure preparation.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00304/ full#supplementary-material

levels of abscisic acid (ABA). J. Am. Soc. Hortic. Sci. 123, 925–930. doi: 10.21273/ JASHS.123.5.925

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ruggiero, Landi, Punzo, Possenti, Van Oosten, Costa, Morelli, Maggio, Grillo and Batelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

#### Regulation of K+ Nutrition in Plants

*Paula Ragel1,2 , Natalia Raddatz1 , Eduardo O. Leidi3 , Francisco J. Quintero1 and José M. Pardo1 \**

*1Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Seville, Spain, 2Centre for Organismal Studies, Universität Heidelberg, Heidelberg, Germany, 3Instituto de Recursos Naturales y Agrobiologia de Sevilla, Consejo Superior de Investigaciones Cientificas, Seville, Spain*

Modern agriculture relies on mineral fertilization. Unlike other major macronutrients, potassium (K+ ) is not incorporated into organic matter but remains as soluble ion in the cell sap contributing up to 10% of the dry organic matter. Consequently, K+ constitutes a chief osmoticum to drive cellular expansion and organ movements, such as stomata aperture. Moreover, K+ transport is critical for the control of cytoplasmic and luminal pH in endosomes, regulation of membrane potential, and enzyme activity. Not surprisingly, plants have evolved a large ensemble of K+ transporters with defined functions in nutrient uptake by roots, storage in vacuoles, and ion translocation between tissues and organs. This review describes critical transport proteins governing K+ nutrition, their regulation, and coordinated activity, and summarizes our current understanding of signaling pathways activated by K+ starvation.

*Edited by:*

*Lam-Son Tran, RIKEN, Japan*

#### *Reviewed by:*

*Manuel Nieves-Cordones, Center for Edaphology and Applied Biology of Segura, Spanish National Research Council (CSIC), Spain Fernando Aleman, The Scripps Research Institute, United States*

#### *\*Correspondence:*

*José M. Pardo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science*

*Received: 07 December 2018 Accepted: 20 February 2019 Published: 20 March 2019*

#### *Citation:*

*Ragel P, Raddatz N, Leidi EO, Quintero FJ and Pardo JM (2019) Regulation of K+ Nutrition in Plants. Front. Plant Sci. 10:281. doi: 10.3389/fpls.2019.00281*

Keywords: plant nutrition, potassium, nitrate, regulation, long-distance transport

# INTRODUCTION

Potassium (K+ ) is of paramount importance in plant cell physiology. K+ is an essential macronutrient that fulfills critical functions related to enzyme activation, osmotic adjustment, turgor generation, cell expansion, regulation of membrane electric potential, and pH homeostasis (Hawkesford et al., 2012). While the K+ concentration in the soil solution may vary widely from 0.01 to 20 mM, plant cells maintain a relatively constant concentration of 80–100 mM in the cytoplasm (Rodriguez-Navarro, 2000). Moreover, plants accumulate large amounts of K+ in their vacuoles, surpassing purely nutritional requirements. Hence, K+ is the most abundant cation in plant cells, comprising up to 10% of plant dry weight and often exceeding the ca. 2% that supports near-maximal growth rates (White and Karley, 2010). There is a steep curvilinear relationship between the tissue concentration of K+ and plant growth, from which a critical concentration of K+ supporting 90% of maximum yield can be determined. Above this concentration, growth has no correlation with the increased K+ content, but at lower K+ concentrations, growth declines rapidly. Consequently, K+ fertilization is common practice in modern agriculture and about 40–60% of crop yields are attributable to commercial fertilizer use (Stewart et al., 2005). However, agricultural fertilization is far from being fine-tuned with nutritional requirements.

K+ is taken up from the soil solution by root epidermal and cortical cells. Once K+ is inside the root symplast, it may be stored in vacuoles, where it fulfills osmotic functions, or is transported to the shoot *via* xylem (Pardo and Rubio, 2011). In turn, shoot cells may also supply stored K+ for redistribution *via* phloem. In this transit from the soil to the different

**97**

plant organs, K+ crosses various cell membranes through K+ specific transport systems (**Figure 1**). Coordinated operation of the different transport systems within the plant to secure K+ uptake from the soil and delivery to the different plant organs requires complex K+ sensing and signaling mechanisms. Because of the extraordinary diversity of K+ transporters in plant cells and the physiological and developmental processes in which they are involved, this review is focused on the molecular mechanisms mediating K+ uptake and release at the plasma membrane level, with an emphasis on K+ absorption from the soil and distribution throughout the plant due to the relevance of these processes in plant nutrition. Storage of K+ into vacuoles is treated only briefly and readers are referred to other comprehensive reviews describing transport systems operating at the tonoplast (Martinoia et al., 2012; Ahmad and Maathuis, 2014; Eisenach and De Angeli, 2017; Martinoia, 2018). Last, because of the extensive interactions of nitrogen and K+ in plant mineral nutrition, we summarize the coordinated regulation of NO3 − and K+ uptake and long-distance transport in *Arabidopsis*.

Uptake and distribution of K+ in plant cells is carried out by a variety of transporter proteins categorized into several families with varied structures and transport mechanisms that comprise the channel families *Shaker*-like voltage-dependent, the tandem-pore (TPK), and the two-pore channels (TPC) (Hedrich, 2012), the carrier-like families KT/HAK/KUP (Nieves-Cordones et al., 2014a; Li et al., 2018), HKT uniporters and symporters (Hamamoto et al., 2015), and cation-proton antiporters (CPA). The CPA family is the largest one and includes the NHX, CHX, and KEA antiporters (Sze and Chanroj, 2018). In this review, we describe the structure and diversity of the main K+ transporter families whose members contribute substantially to K+ nutrition. Other proteins with uncertain roles or descriptions of transport activities without candidate proteins have been omitted.

#### TRANSPORT PROTEIN FAMILIES INVOLVED IN K+ NUTRITION

#### K+ -Selective Channels

The first K+ transporter with a role in nutrient uptake was the *Shaker*-like, voltage-gated, and K+ -selective channel AKT1 (Hirsch et al., 1998). Although voltage-gated (VG) channels of plants are phylogenetically related to animal *Shaker* channels, they are distinct and include additional functional domains (Jegla et al., 2018). The basic architecture of VG channels consists of four α-subunits surrounding a central aqueous pore for K+ permeation. Each subunit contains six transmembrane segments, named S1–S6, which can be divided into two different modules: the first four α-helices form a voltage-sensor domain that contains multiple positively charged residues that moves within the membrane in response to voltage. This movement is directly coupled to the opening or closing of the channel. The segments S5, S6, and the pore loop, form the pore domain, named P, where each of the four subunits contributes equally to the permeation pathway. Moreover, plant α-subunits have a long C-terminal region constituting more than half of the protein (**Figure 2**). This cytosolic tail includes several functional domains: (1) a linker region (C-linker) proximal to the pore

FIGURE 1 | Transporters involved in K+ uptake by roots and inter-organ partition. HAK5, AKT1, and non-selective cyclic nucleotide-gated cation channels (CNGC) all contribute to K+ nutrition, albeit at different ranges of substrate concentrations, from low- to high-availability, respectively. K+ efflux through the outward-rectifying GORK channel facilitates the fine-tuning of plasma membrane electrical potential, and allows repolarization under circumstances that promote depolarization, such as salinity stress. In the root stele, the outward-rectifying SKOR channel releases K+ into the xylem vessels for nutrient delivery to the shoots. The nitrate transporter NRT1.5 facilitates K+ uploading into the xylem either by electrical coupling with other K+ -selective transporters or directly acting as K+ /H+ antiporter. In aerial tissues, an array of K+ -influx channels and KT/HAK/KUP carriers allow the uptake of the incoming K+ into green cells. K+ is stored inside vacuoles by NHX exchangers and released back to the cytosol by TPK and TPC1 channels, and possibly also by KT/HAK/KUP carriers at the tonoplast (the vacuole in root cells is omitted for simplicity). The plasma membrane outward K+ channel AKT2 releases K+ into the phloem for returning K+ to the root and to facilitate the uploading of photosynthates into the phloem sap.

that transduces conformational changes that gate the channel and that may also determine the target membrane (Nieves-Cordones et al., 2014b; Jegla et al., 2018); (2) a conserved and essential cyclic nucleotide-binding homology domain (CNBHD) whose function is not the binding of cNMP but to mediate the interactions between subunits within the channel tetramer; (3) an ankyrin domain (found in only six out of the nine *Arabidopsis* VG channels), which may mediate the binding of interacting proteins (Michaely and Bennett, 1992); and (4) a distal KT/KHA domain rich in hydrophobic and acidic residues, that is unique to plant K+ channels, and is involved in channel tetramerization and clustering at the membrane (Daram et al., 1997; Ehrhardt et al., 1997; Zimmermann et al., 2001; Dreyer et al., 2004).

Plant voltage-gated K+ channels are divided into three subfamilies regarding their response to the membrane potential (Dreyer and Uozumi, 2011): (1) Inward-rectifying (Kin) channels that in *Arabidopsis* include AKT1, AKT6, KAT1, and KAT2; they open at hyperpolarized membrane potentials allowing the uptake of K+ . (2) Outward-rectifying (Kout) channels that mediate K+ release because they open at depolarized membrane potentials; this group is composed of SKOR and GORK channels. (3) Weakly rectifying (Kweak) channels that can mediate both K+ uptake and release, and whose *Arabidopsis* representative is AKT2. In addition, the *Arabidopsis* KC1 (KAT3) is an electrically silent *Shaker*-like protein that interacts with and regulates functionality of the Kin channels AKT1, KAT1, KAT2, and AKT2, but not the Kout channels (Jeanguenin et al., 2011). This interaction negatively shifts the activation threshold of Kin channels and decreases the macroscopic inward conductance compared to that of homomeric channels (Dreyer et al., 1997; Duby et al., 2008; Geiger et al., 2009). Heteromerization of different subunits of Kin channels is of great importance to increase the functional diversity and regulation of different cell types (Dreyer et al., 1997; Véry and Sentenac, 2003; Xicluna et al., 2007; Jeanguenin et al., 2008; Lebaudy et al., 2008; Lebaudy et al., 2010). Although this behavior has also been suggested for outward-rectifier (Kout) channels, heteromerization has been reported only among subunits of Kin or Kout, preventing formation of heteromeric structures between the two subunit types (Dreyer et al., 2004).

#### K+ -Uptake Carriers

Proteins of the KT/HAK/KUP family are present in plants, fungi, bacteria, and even viruses (Greiner et al., 2011; Santa-Maria et al., 2018), and they are often associated with K+ transport across membranes and K+ supply. In bacterial genomes, K+ carriers of this family are encoded by single-copy genes named *kup*. In *Escherichia coli*, kup is a constitutive low-affinity uptake system that operates as K+ -H+ symporter (Zakharyan and Trchounian, 2001). In fungi, the homologous proteins are encoded by *HAK1*-like genes present as one- or two-copy in most species. In contrast to bacterial *kup*, fungal *HAK* genes are strongly induced by K+ starvation and the encoded proteins mediate high-affinity K+ transport (Benito et al., 2011). In plants, these transporters are known as KT, HAK, or KUP (KT/HAK/KUP family) and they are represented by multiple genes in their genomes. Members of this family have been widely associated with high-affinity K+ uptake from the soil, while others may function in both low-affinity and/or highaffinity transport (Luan et al., 2009; Very et al., 2014) and other roles related, for example, to K+ translocation, control of water movement at the plant level, salt tolerance, osmotic/ drought responses, transport of other alkali cations, and developmental processes in plants, such as root hair growth and auxin distribution (Li et al., 2018; Santa-Maria et al., 2018). These diverse functions of KT/HAK/KUP transporters may all result from their critical roles in cellular K+ homeostasis. *KT/HAK/KUP* genes are not present in animal cells, what could indicate that they are crucial for K+ transport in organisms facing external solutions with fluctuating and very low K+ concentrations, often in the μM range (Ashley et al., 2006). Based on the present knowledge, *KT/HAK/KUP* genes are present in all plant genomes, which contrasts with that in the other kingdoms, where they are only present in certain species (Grabov, 2007; Greiner et al., 2011). This difference may reflect the importance of these transporters for the plant's way of life.

KT/HAK/KUP transporters of land plants are classified according to their sequence homology into six clusters or clades (I–VI), with clade VI including only members of bryophytes (Santa-Maria et al., 2018). Phylogenetic analysis shows that all KT/HAK/KUPs from algae diverge from land plant clades, suggesting that the diversification into these groups took place after the colonization of land by green organisms (Santa-Maria et al., 2018). The KT/HAK/KUP group from angiosperms displays a high and rather variable number of members in the different plant species genomes that have been sequenced so far. For instance, there are 13 genes in *Arabidopsis*, 16 in peach, 17 in grapevine, 20 in *Medicago*, 21 in *Cassava*, 27 in rice, maize, and *Brachypodium,* and 57 in *Panicum virgatum* (Song et al., 2015; Nieves-Cordones et al., 2016a,c; Ou et al., 2018). Members of KT/HAK/KUP family in angiosperm are classified among clades I–V (Nieves-Cordones et al., 2016c).

The KT/HAK/KUP transporters involved in K+ uptake from the soil are clustered into a distinct subgroup of clade I, termed Ia (Nieves-Cordones et al., 2016c) and that we call herein HAK1-like transporters by analogy with the fungal counterparts. This subgroup includes barley HvHAK1 (Santa-María et al., 1997; Gierth and Mäser, 2007), *Arabidopsis* AtHAK5 (Rubio et al., 2000; Gierth et al., 2005), rice OsHAK1 and OsHAK5 (Bañuelos et al., 2002), pepper CaHAK1 (Martinez-Cordero et al., 2004), tomato LeHAK5 (Nieves-Cordones et al., 2007), and *Thellungiella* ThHAK5 (Alemán et al., 2009b). High-affinity K+ transport has been demonstrated for all the HAK1-like transporters in heterologous expression systems (Nieves-Cordones et al., 2014a). On the other hand, AtKUP7, belonging to clade V, could be involved in K+ uptake from low to moderate external K+ concentrations (Han et al., 2016), and thus the participation in K+ uptake from soil of proteins from different clades should not be discarded. Interestingly, other members of clade I could be related with K+ uptake by cells from specialized tissues. DmHAK5 from Venus flytraps is implicated in the uptake of K+ released from the digested prey in the bi-lobed capture organ (Scherzer et al., 2015), whereas the quinoa CqHAK5-like drives K+ influx into cells of the leaf salt bladders to contribute to the osmotic balance of the cytosol against the osmotic pressure of the salt-containing vacuoles (Bohm et al., 2018).

KT/HAK/KUP transporters are phylogenetically related to the superfamily of acid-polyamine-organocation (APC) transporters that comprises secondary active transport proteins responsible for uniport, symport, and antiport of a wide range of substrates (Vastermark et al., 2014). Taking as template crystal structures of prokaryotic APC transporters, computational 3D modeling of AtKUP7 (Ahn et al., 2004; Al-Younis et al., 2015; Santa-Maria et al., 2018), AtKUP4/TRH1 (Daras et al., 2015), OsHAK1 (Rai et al., 2017), AtKUP1/TRH1 (Santa-Maria et al., 2018), AtHAK5 (Santa-Maria et al., 2018), and HvHAK1 (Santa-Maria et al., 2018) has been reported. The structural models (**Figure 2**) show the presence of common attributes among all of them: (1) a hydrophobic core containing 10–14 transmembrane (TM) segments; and (2) three cytosolic domains—the N- and C-termini and a region containing approximately 70 residues situated between second and third TMs (loop II–III). Although, the structure of the pore region has not been described yet, several works have analyzed the effect of mutations on the function of these transporters (Santa-Maria et al., 2018). So far, results indicated that several parts of the protein may contribute to setting the Vmax of the transporter and that the region including from N-terminus to loop II–III may contribute in determining its Km. Furthermore, sequence alignments show that, although there is not extensive sequence conservation, 40 amino acid residues are conserved in exactly the same position in all the eukaryotic HAK transporters and in the Kup bacterial transporters (Rodriguez-Navarro, 2000). Six of these conserved residues are included in a highly conserved motif in the first transmembrane domain whose consensus sequence is **G**VVY**GD**LGTS**PLY** (the amino acids conserved in all proteins are in bold) (Rodriguez-Navarro, 2000). A helical-wheel representation of this transmembrane fragment locates three glycine residues on the same side of the helix, which in the case of a tetrameric structure may operate as a substrate selectivity filter analogous to the GXGYGD motif highly conserved in K+ channels. Regarding this, it has been suggested that AtKUP4/TRH1 may form homodimers (Daras et al., 2015), likely involving the interaction between C-terminus domains and less likely between loops II–III.

Contrary to VG channels that are all targeted to the plasma membrane, KT/HAK/KUP transporters have been reported in different subcellular compartments (**Table 1**). The majority of the characterized transporters of the KT/HAK/KUP family are located in the plasma membrane, although not all of them are involved in K+ nutrition. For instance, AtKUP4/TRH1 seems to participate in auxin transport related with root gravitropism and root hair development (Rigas et al., 2013), whereas AtKUP6 acts in lateral root initiation and development in the auxin and ABA crosstalk signaling pathways (Osakabe et al., 2013).

TABLE 1 | Sub-cellular location of selected *Arabidopsis*, barley, rice, and *Physcomitrella patens* KT/HAK/KUP transporters.

#### HKT Proteins

The ample repertoire of transporters encoded in the genome of plants includes proteins that are collectively known as High affinity K+ Transporters (HKTs; **Figure 2**) despite the fact that these proteins facilitate Na+ -selective uniport or Na+ -K+ symport with a channel-like activity (Benito et al., 2014). Phylogenetic and functional analyses distinguished two HKT subfamilies (Platten et al., 2006). Members of subfamily I (HKT1) are ubiquitous in plants, Na+ -selective, and mostly involved in Na+ recirculation through vascular tissues, as best exemplified by AtHKT1;1 (Sunarpi et al., 2005). Members of subfamily II (HKT2) have been found only in monocotyledonous species. Although they are all K+ -permeable, mechanistically HKT2s can operate as either Na+ -K+ symporters or K+ -selective uniporters [reviewed by Benito et al. (2014)]. HKT2-like proteins of cereals have been involved in K+ nutrition.

#### K+ UPTAKE BY ROOTS

The uptake of K+ by roots (often measured with rubidium as tracer) exhibits a biphasic kinetics in response to increasing external concentrations corresponding to high- and low-affinity transport systems, which work at low (<1 mM) and high (>1 mM) external K+ concentrations respectively (Epstein et al., 1963; Gierth and Mäser, 2007). At high concentration in the soil solution, K+ crosses the membrane mostly through channels. The channels simply give a path for the ions allowing them to move down the electrochemical gradient. At low K+ concentration, active transporter systems are needed in order to pull K+ inside the cell against its electrochemical gradient. However, studies in several plant species have shown that channels may be involved in K+ uptake in the high-affinity range of K+

concentrations (Rubio et al., 2010) as long as the membrane is sufficiently hyperpolarized, i.e. highly electronegative inside (Hirsch et al., 1998; Spalding et al., 1999; Gierth and Mäser, 2007; Rubio et al., 2010).

The sensitivity to NH4 + is an important characteristic of highaffinity K+ uptake mediated by carriers that has been used as a tool for the identification of additional high-affinity transport systems (Santa-María et al., 1997; Nieves-Cordones et al., 2007). NH4 + -sensitive and -insensitive components of high-affinity K+ uptake have been identified in *Arabidopsis* (Spalding et al., 1999), barley (Santa-Maria et al., 2000), pepper (Martinez-Cordero et al., 2005), and rice (Bañuelos et al., 2002; Chen et al., 2015). Results indicate that the NH4 + -sensitive component of K+ uptake is likely mediated by KT/HAK/KUP transporters (HAK1-like transporters), whereas inward-rectifier K+ channels (AKT1-like channels) constitute the NH4 + -insensitive pathway (Santa-Maria et al., 2000; Nieves-Cordones et al., 2014a). Together, AKT1-like channels and HAK1-like transporters are now thought to constitute the main systems for K+ uptake in plants under low-K+ concentrations (**Table 2**). However, the NH4 + -sensitive and -insensitive pathways appear to contribute differently to highaffinity K+ uptake depending on the plant species and the ionic external concentration of transported substrates, mainly K+ , NH4 + , and Na+ (Aleman et al., 2011; Nieves-Cordones et al., 2016c).

Among the inward-rectifying K+ channels of *Arabidopsis*, only AKT1 and AtKC1 are abundantly expressed in root tissues (Reintanz et al., 2002). AtKC1 expressed alone remains in the endoplasmic reticulum, but it can be recruited to the plasma membrane to regulate AKT1 activity (Duby et al., 2008; Geiger et al., 2009; Honsbein et al., 2009; Wang et al., 2010). In addition, AKT1 is positively regulated by the protein kinase complex comprising the kinase CIPK23 and one of the two alternative calciumdependent regulatory subunits CBL1 and CBL9 (Li et al., 2006;

TABLE 2 | Comparison of AKT1 channels and HAK1/HAK5 transporters from *Arabidopsis* and rice working at different ranges of external K+ concentrations. The uptake systems working in addition to AKT1 and HAK1/HAK5 likely include CHX exchangers (Zhao et al., 2008) and cyclic nucleotide-gated channels (CNGC) that may contribute to K+ absorption when the external K+ concentration is sufficiently high (Caballero et al., 2012).

Xu et al., 2006). AKT1 possesses an intrinsic K+ sensor reducing channel conductance at submillimolar external K+ concentrations. Despite this K+ sensor, upon activation by the CIPK/CBL complex at low external K+ , the homomeric AKT1 channels open at voltages positive of *E*K, a condition potentially resulting in cellular K+ leakage (Geiger et al., 2009). Incorporation of the *At*KC1 subunit into the channel complex, however, shifts the voltage dependence of AKT1 toward more negative potentials (ca. −70 mV) to prevent K+ loss (Geiger et al., 2009; Wang et al., 2010; Wang et al., 2016). In other words, AKT1/KC1 heteromerization renders the channel more efficient at blocking K+ permeation in the outward direction. The physical interaction of the CIPK23/CBL1 complex is specific for AKT1 channels and does not involve the AtKC1 subunit. The gain-of-function mutation *AtKC1-D* (G322D substitution in transmembrane S6) was recovered in the *cipk23* mutant background. *AtKC1-D* enhanced the inhibition of AKT1 channel activity and restricted K+ leakage through AKT1 under low-K+ conditions, thereby increasing the tolerance to nutrient stress (Wang et al., 2016). Although the double mutant *akt1 KC1-D* was sensitive to low-K+ , indicating that KC1-D action is through AKT1, an additional indirect effect of mutation KC1-D through HAK5 cannot be ruled out. By inhibiting AKT1 and shifting its voltage dependence toward a more negative direction, the plasma membrane could become hyperpolarized in the *KC1-D* mutant, thereby enhancing the expression and activity of HAK5 and improving net K+ uptake.

Several mechanisms for AKT1 deactivation have been proposed. The PP2C-type protein phosphatase AIP1 interacts with and inactivates the AKT1 channel, counteracting the activation by CIPK23 in oocytes (Lee et al., 2007). In principle, these findings are evidence of a phosphorylation/dephosphorylation switch that regulates AKT1 channel activity, but it should be noted that no phosphorylation of AKT1 by CIPK23 and dephosphorylation by AIP1 has been demonstrated conclusively (Hashimoto et al., 2012). Instead, four components, CIPKs, CBLs, PP2Cs, and AKT1, appear to interact mutually and form a molecular complex whose specific composition could ultimately regulate channel activity (Lan et al., 2011). In this model, PP2C phosphatases interact with the kinase domain of CIPKs to counteract kinasemediated activation of AKT1. Upon calcium signaling, CBLs interact with PPC2C to inhibit their phosphatase activity while simultaneously activating the partnering CIPKs. On the other hand, CBL10, a regulatory subunit of CIPK24/SOS2 but not of CIPK23, also interacts directly with AKT1 and negatively modulates AKT1 activity by competing with CIPK23 to bind AKT1 (Ren et al., 2013). Since CBL10 function is related to salinity stress rather than to mineral nutrition (Kim et al., 2007; Quan et al., 2007; Lin et al., 2009), this cross-regulation may constitute a mechanism to prevent salinity-induced K+ loss though AKT1. In line with this, the nitric oxide (NO) that accumulates under salinity stress also inhibits the K+ uptake mediated by AKT1. The link is indirect since NO triggered the accumulation of pyridoxal 5′-phosphate (PLP), an active form of vitamin B6, that in turn repressed the activity of AKT1 in *Xenopus* oocytes and *Arabidopsis* root protoplasts (Xia et al., 2014).

In *Arabidopsis*, the voltage-gated channel GORK (guard cell outward-rectifying K+ ) is the major outward-rectifying K+ channel in guard cells where it contributes to K+ efflux for decreasing turgor and stomatal closure (Ache et al., 2000; Hosy et al., 2003). In addition, GORK is expressed in root outer cell layers (epidermal, root hairs, and cortex) of *Arabidopsis* and thus GORK is considered a major pathway for stressinduced K+ leakage from root cells, e.g. by exposure of roots to high salt (Ivashikina et al., 2001; Demidchik et al., 2010; Demidchik et al., 2014). Production of hydroxyl radicals (HO˙) in salinized roots stimulates a dramatic K+ efflux mediated by GORK from root cells (Demidchik et al., 2010). The oxidative and salt stresses cause programmed cell death (PCD) and collapse membrane potential in root cells of *Arabidopsis thaliana* in a K+ -dependent manner. Accordingly, the *Arabidopsis gork1-1* mutant showed no K+ outwardly directed currents in response to HO˙. Besides, after exposure to high NaCl levels, the mutant *gork1-1* displayed lower activity of proteases and endonucleases for PCD, which in the wild type was dramatically enhanced by K+ loss in root cells (Demidchik et al., 2010).

Both the expression level and channel activity of GORK are significantly upregulated by increasing levels of the abscisic acid (ABA) and jasmonate. Stimuli that elevated endogenous ABA concentrations, e.g. drought, osmotic stress, or cold, led to the up-regulation of *GORK* transcripts (Becker et al., 2003; Suhita, 2004) while treatment with salicylic acid inhibited the presence of active GORK channels and improved salinity tolerance through prevention of K+ efflux. Recent studies demonstrated that calcium-dependent protein kinase 21 (CPK21) phosphorylated GORK and suggested that 14-3-3 proteins control GORK activity through binding with CPK21. This kinase phosphorylates three amino acid residues in the C-terminus of GORK, T344, S518, and S649. Binding of 14-3-3 to CPK21 strongly stimulated its kinase activity and increased Ragel et al. Regulation of K+ Nutrition in Plants

GORK phosphorylation (van Kleeff et al., 2018). On the other hand, the phosphatase AtPP2CA interacts physically with GORK inhibiting its current (Lefoulon et al., 2016). Thus, AtPP2CA could have an antagonist role to CPK21 on the regulation of GORK (van Kleeff et al., 2018). These results imply that the salinity-induced membrane depolarization together with the Ca2+- and CPK21-dependent phosphorylation act together to activate GORK and to repolarize the plasma membrane by means of releasing part of the cytosolic K+ . Moreover, the peak of the salt-induced K+ -efflux in the *aha2* mutant, devoid of a major isoform of the plasma membrane H+ -ATPase, was stronger and more sustained than in the wild-type, suggesting that H+ -pumps take over membrane repolarization after the initial K+ -loss to re-enact K+ uptake (van Kleeff et al., 2018). Recently, Saponaro et al. (2017) showed that 14-3-3 proteins are also capable of modulating KAT1, although in this case 14-3-3 bound directly to the KAT1 C-terminus affecting both the voltage dependency of the channel and the number of channel molecules in the membrane (Sottocornola et al., 2008).

K+ -H+ symport has long been considered the likely catalytic mechanism of plant KT/HAK/KUP transporters based on the demonstration that K+ -H+ symport operates in K+ -starved *Neurospora crassa* and on thermodynamical considerations regarding the steep K+ gradient that KT/HAK/KUP proteins are able to achieve across cell membranes that exceeds what could be reached by coupling the K+ uptake to the membrane potential solely (Rodriguez-Navarro, 2000). Until recently, efforts to express plant KT/HAK/KUP proteins in *Xenopus* oocytes to measure K+ currents had failed, but work with the DmHAK5 transporter from Venus flytraps showed that co-expression of the corresponding cRNA with that of CBL9/CIPK23 (but not DmHAK5 alone) generated inward K+ and Rb+ currents in *Xenopus* oocytes that were stimulated by low external pH (Scherzer et al., 2015). Moreover, salt bladders of the halophyte *Chenopodium quinoa* that accumulate salts to very high concentrations express a HAK-like activity driving high-affinity and selective K+ uptake that was dependent on acidic external pH and by the CIPK23/CBL1 kinase module of *Arabidopsis* (Bohm et al., 2018). Electrophysiological recordings in rice roots showed that the activity of OsHAK1 was strongly electrogenic and depolarizing. Plots of the OsHAK1-dependent K+ -induced membrane depolarization had a slope of 29 mV per decade of external K+ concentration, suggesting the co-transport of two monovalent cations (a 59 mV slope is to be expected from an uniprot transport moving only single K+ ions) (Nieves-Cordones et al., 2017). Together, these data strongly suggest that plant KT/ HAK/KUP proteins operate as K+ -H+ symporters. Residues involved in K+ binding and/or transport have not been identified; however, mutant proteins with residue substitutions of members of the KT/HAK/KUP family have been described as showing modified affinity for K+ , Na+ , and/or Cs+ , or increased Vmax (Aleman et al., 2014).

HAK1-like transporters are subject to complex transcriptional and post-translational regulations, although studies have been carried out almost exclusively in *Arabidopsis* AtHAK5 (Jung et al., 2009; Rubio et al., 2014; Ragel et al., 2015). Under any stress conditions that directly affect K+ acquisition, such as K+ deprivation or salinity, high-affinity K+ uptake systems should be transcriptionally or post-translationally activated in order to maintain the K+ supply and K+ /Na+ homeostasis. Accordingly, all characterized HAK1-like transporters exhibit low expression levels in roots under control conditions, are highly up-regulated upon K+ deprivation and rapidly down-regulated when K+ is resupplied (reviewed by (Li et al., 2018)). Furthermore, it has been commonly observed that other ions, particularly NH4 + , NO3 − , Na+ , and Pi, also regulate the expression of *HAK1*-like genes and not always in the same way (Nieves-Cordones et al., 2019). For example, NH4 + reduces the transcriptional induction by K+ starvation of the pepper *CaHAK1* (Martinez-Cordero et al., 2005) and *Arabidopsis AtHAK5* (Qi et al., 2008), but enhances the expression of *LeHAK5* in tomato (Nieves-Cordones et al., 2007). The presence of NaCl prevents the induction of *LeHAK5* by K+ starvation (Nieves-Cordones et al., 2007), but provokes a strong and transient up-regulation of *HvHAK1* (Fulgenzi et al., 2008). Thus, the *Arabidopsis* model cannot be completely extended to other plant species, crops among them. In contrast to HAK1-like transporters, KT/HAK/KUP proteins belonging to clusters II–V show diverse expression patterns and most of them do not exhibit transcriptional regulation in response to K+ deficiency (Ahn et al., 2004; Li et al., 2018). For example, *AtKUP7* (cluster V, plasma membrane) transcript is not induced by low-K+ (Han et al., 2016) and *AtKUP12* (cluster III, chloroplast) is down-regulated after K+ resupply (Armengaud et al., 2004).

Regarding the transcriptional regulation of genes encoding HAK1-like transporters, it has been shown that the effect of the nutrient deficiency and salt stresses on transcriptional expression of *AtHAK5* and *LeHAK5* is associated with changes in the root cell membrane potentials (Nieves-Cordones et al., 2008; Rubio et al., 2014); the hyperpolarization of the plasma membrane of root cells induces transcription of both genes. Supporting this, *ThHAK5* of *Thellungiella halophila* (salt cress, a.k.a. *Eutrema salsuginea*) is expressed to higher levels than *AtHAK5* under salt stress, while roots of *T. halophila* maintained a more negative membrane potential than *Arabidopsis* roots (Volkov and Amtmann, 2006; Alemán et al., 2009b; Rubio et al., 2014). Besides membrane hyperpolarization, the expression of *AtHAK5* is also induced, under K+ -limiting conditions, as result of signaling cascades that involve ROS production, phytohormones, and transcription factors. Low-K+ stress, alike other nutrient-deprived conditions, promotes an increase of ethylene that positively regulates ROS production in roots (Shin and Schachtman, 2004; Jung et al., 2009). Roots deprived of K+ induce the expression of genes involved in ethylene biosynthesis and signaling, and in ROS metabolism, promoting two-fold higher levels of ethylene and the increase in hydrogen peroxide (H2O2) concentrations. Both ethylene and ROS give rise to enhanced transcription of *HAK5* in *Arabidopsis* and tomato (Rodenas et al., 2018). In *Arabidopsis*, H2O2 produced by the NADPH oxidase RHD2/RbohC regulates the expression of *AtHAK5* in response to K+ deficiency (Shin and Schachtman, 2004) and it has been proposed that peroxidase RCI3 (Rare Cold Inducible gene 3) contributes to ROS production during *Arabidopsis* root response to K+ deficiency (Kim et al., 2010). In the case of ethylene-induced *AtHAK5* transcription, the intermediaries in ethylene signaling CTR1 (Constitutive Triple Response (1) and EIN2 (Ethylene Insensitive (2) are partially involved. Results also suggest the existence of other signaling pathways or an EIN2-independent ethylene route that may play an important role in low-K+ signaling (Jung et al., 2009). Genetic hierarchy indicates that ethylene signaling acts upstream of ROS when plants are deprived of K+ (Jung et al., 2009). Nevertheless, it has also been speculated that a positive feedback may stimulate ethylene-induced ROS production (Wang et al., 2002). Other hormones have been shown to be involved in K+ deprivation signaling and response, for instance jasmonic acid (Armengaud et al., 2004), auxin (Jung et al., 2009; Hong et al., 2013), ABA (Kim et al., 2010), cytokinins (Nam et al., 2012), and gibberellins through DELLA proteins (Oliferuk et al., 2017). Cytokinins are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate, and sulfate transporters. Cytokinin content decreases under K+ -starved conditions, and cytokinin-deficient mutants, under same conditions, display enhanced accumulation of both ROS and *AtHAK5* transcripts (Nam et al., 2012). By contrast, cytokinin-receptor mutants lost the responsiveness to low-K+ , including ROS accumulation and root hair growth. Interestingly, the cytokinin/ethylene ratio is positively correlated with tomato shoot biomass, suggesting that the balance between both hormones is important in determining the plant vigor at low-K+ supply, but with an inverse role in tomato compared to *Arabidopsis*, where cytokinin/ethylene ratio was negatively correlated with tolerance to K+ deprivation (Jung et al., 2009; Nam et al., 2012).

In addition to low nutrient conditions, salt stress (and presumably other abiotic stresses) results in modifications of *AtHAK5* expression or the low-K+ response. Mild salt stress does not induce *AtHAK5* expression but its expression levels gradually increased following an increase in NaCl concentrations (Ahn et al., 2004; Hong et al., 2013). This suggests that plants may recognize high Na+ levels as K+ deprivation. However, the induction of *AtHAK5* expression by low K+ was suppressed by salt stress in *Arabidopsis* (Nieves-Cordones et al., 2010), but not in *T. halophila* (Alemán et al., 2009a). As discussed above, under salt stress conditions, *T. halophila* registers a more negative root membrane potential than *A. thaliana* (Volkov and Amtmann, 2006), which may explain the expression of *ThHAK5* under these conditions (Alemán et al., 2009a).

In recent years, several transcription factors (TFs), and their target sequences, have been identified in the *AtHAK5* promoter. Among them, ARF2 (Auxin Response Factor 2) is the only one described so far to work as negative regulator of *AtHAK5* transcription (Zhao et al., 2016). Interestingly, ARF2 has been found to be involved in many phytohormone-signaling pathways, but it seems not to participate in auxin signaling. Under K+ sufficient conditions, channel-mediated K+ uptake would be energetically more favorable than symport through AtHAK5, and hence *AtHAK5* should be shut down (Zhao et al., 2016). In those conditions, ARF2 binds to the auxin-responsive elements (AuxREs) within the *AtHAK5* promoter and represses transcription. When plants are subjected to low-K+ stress, ARF2 is rapidly phosphorylated by an unknown kinase and loses DNA binding activity. ARF2 is removed from the *AtHAK5*

promoter, which relieves the repression on *AtHAK5* transcription. In turn, other TFs bind to the *AtHAK5* promoter and activate its transcription. These TFs up-regulating *AtHAK5* expression under K+ starvation include RAP2.11, which binds to the ethylene-responsive element (ERE) and the GCC-box of the *AtHAK5* promoter, and whose expression is stimulated by ethylene and ROS, alike *AtHAK5* (Kim et al., 2012). TFs DDF2, JLO, bHLH121, and TFII\_A also interact with the upstream region of *AtHAK5*, but the specific binding motif for each of them has not been identified yet (Hong et al., 2013). All of these transcription factors are sufficient to activate *AtHAK5* expression in heterologous systems, but none of them is absolutely required. When K+ is resupplied, ARF2 becomes dephosphorylated again and represses *AtHAK5* expression (Zhao et al., 2016). Thus, it is apparent that regulation of the activity of TFs acting on *AtHAK5* transcription (positively or negatively) is necessary to determine cooperatively the accumulation of the corresponding transcripts (Santa-Maria et al., 2018).

Although a general nutrient deprivation stimulus is sufficient for the transcriptional activation of *AtHAK5* and *LeHAK5* genes, a reduction of internal K+ is required for the induction of a functional HAK5-mediated high-affinity K+ uptake in *Arabidopsis* and tomato roots (Rubio et al., 2014), suggesting the existence of post-transcriptional regulation *in planta*. Recently, it was shown that activation of high-affinity K+ uptake mediated by AtHAK5 (Ragel et al., 2015), DmHAK5 from Venus flytraps (Scherzer et al., 2015), and CqHAK from quinoa (Bohm et al., 2018) is mediated by the CBL-interacting protein kinase (CIPK)/ calcineurin B-like protein (CBL) complex comprising CIPK23 and CBL1/9 proteins of *Arabidopsis*. Notably, this CIPK23/CBL1,9 module also activates AKT1 channel, that together with AtHAK5 constitutes the main K+ uptake pathway in *Arabidopsis* roots (Xu et al., 2006; Lee et al., 2007). Both the protein kinase AtCIPK23 and the Ca2+ sensor AtCBL1 are necessary and sufficient for activation of the high-affinity K+ transporter AtHAK5 in yeast (Ragel et al., 2015). Besides AtCBL1, other CBLs (AtCBL8/9/10) are able to bind AtCIPK23 and activate AtHAK5 to complement K-uptake defective yeast growth. The reduction in the K+ concentration produces a specific Ca2+ signature in the cytosol (**Figure 3**) (Behera et al., 2017) that would be recorded by AtCBL1, promoting CIPK23/CBL1 complex formation, and the activation of AtHAK5 by phosphorylation at the cytosolic N-terminus (Ragel et al., 2015), in a similar way that was described for AKT1 (Xu et al., 2006; Lee et al., 2007). The enhancement of growth at low-K+ of yeast cells co-expressing AtHAK5, AtCIPK23, and AtCBL1 seems to result from modification of the kinetic properties of the transporter (Km decrease and Vmax increase), likely through the phosphorylation-induced conformational changes of AtHAK5 (Ragel et al., 2015). However, since physical interaction between CIPK23/CBL1 and AtHAK5 is also required for full activation of AtHAK5 in yeast, the trafficking of the transporter to plasma membrane has been proposed as a second mechanism of AtHAK5 regulation by CIPK23/CBL1 complex. Supporting this idea, the AtHAK5 protein was mainly detected in the endoplasmic reticulum of K+ -sufficient plants, while K+ starvation produced an enrichment of AtHAK5 protein in the plasma membrane (Qi et al., 2008).

In heterologous systems, the *Arabidopsis* CIPK23/CBL1,9 complex enabled the activation of various members from clade I of KT/ HAK/KUP transporters, such as pepper CaHAK1 (Ragel et al., 2015) and Venus flytrap DmHAK5 (Scherzer et al., 2015), but not of tomato SlHAK5 or the *Eutrema salsuginea* EsHAK5 (Ragel et al., 2015). These results suggested that the activation mechanism by CIPK23/CBLs complexes is evolutionarily conserved, but not the phosphorylation site and/or the target sequence recognition, which may vary among distant plant species. Accordingly, a quimeric tomato HAK5 protein that contained the 15 first amino acids of CaHAK1 could be activated by CIPK23/CBL1 in yeast (Ragel et al., 2015).

Since the two main contributors to K+ uptake in *Arabidopsis*, AKT1 and HAK5, are regulated by the CIPK23/CBL1,9 complex, the coordinated regulation of these transport systems deserves attention. Under K+ -sufficient conditions, K+ uptake by HAK5 would be energetically more expensive than permeation through the AKT1 channel. The H+ -pumping activity of plasma membrane ATPases, which is used for many secondary transport processes, creates *per se* an electrical charge (negative inside) that suffices to draw significant amounts of K+ into the cytosol. At a regular steady membrane potential of −120 to −180 mV, root epidermal cells could sustain a 100–1,000-fold inward-directed gradient of K+ . However, coupling K+ uptake to H+ influx not only returns H+ to the cytoplasm but also is more depolarizing than simple K+ permeation, which in turn imposes a greater demand on the H+ -pumps and ATP consumption. We speculate that under such conditions of K+ sufficiency, the plasma membrane is not hyperpolarized (or not enough), signaling phytohormones are not produced, and therefore transcription of *HAK5* is not activated. AKT1 would be operational through the physical interaction with CIPK23 (and possibly other CIPKs) (Lee et al., 2007). As the K+ concentration outside decreases, the function of AKT1 becomes increasingly hampered and full activation by the Ca2+ dependent CIPK23/CBL1,9 complex is required to sustain K+ uptake, while the KC1 safeguard prevents K+ leakage through AKT1 (Wang et al., 2016). CIPK23 is known to display different states of activation, depending on factors that affect the activation of CIPKs by upstream kinases (Barajas-Lopez et al., 2018) and CBL binding (Chaves-Sanjuan et al., 2014). Thus, a mild K+ deprivation may produce a partially activated CIPK23 that would be competent for activating AKT1 but not HAK5, whereas severe K+ deprivation leads to *HAK5* transcription and to full activation of CIPK23, which would then be competent for activating HAK5 (Ragel et al., 2015). The *CIPK23* is itself induced transcriptionally by low-K+ stress (Xu et al., 2006), which could also enhance the response to nutritional stress.

The participation of Ca2+ sensors in high-affinity K+ uptake could mechanistically connect K+ starvation with other abiotic stresses, for instance: salinity, water availability, oxygen deficiency (hypoxia) or absence (anoxia), mechanical stress, cold stress, heavy metal stress, and other nutrient deprivations, all sharing cytosolic free Ca2+ as a second messenger (Wilkins et al., 2016). Current thinking is that the specificity of Ca2+ signaling is determined by the amplitude and duration (and possible oscillation) of the cytosolic Ca2+ increase, often referred to as the "calcium signature" that is elicited by the stimulus. K+ deficiency evokes two successive Ca2+ signals in roots exhibiting different spatial and temporal specificity (Behera et al., 2017). The first one is characterized by a transient and fast Ca2+ increase within 1 min in the postmeristematic elongation zone (most prominently in the vascular tissue and endodermis), followed by a Ca2+ return nearly to basal concentrations in <7 min. The second wave (secondary Ca2+

response) occurs after several hours (18 h) as sustained Ca2+ elevation in defined tissues of the elongation and root hair differentiation zones. It has been proposed that this secondary Ca2+ elevation would contribute to long-term adaptation responses (Behera et al., 2017). These differential Ca2+ signatures could also underpin the step-wise activation of CIPK23/CBL1,9 discussed above. Ca2+-sensing proteins with diverse Ca2+ affinities, subcellular localizations, and downstream target specificities may perceive Ca2+ signatures differentially and transduce them into adequate downstream signaling responses (Hashimoto and Kudla, 2011).

Besides the CIPK23/CBL1,9 module, evidence for a second Ca2+-dependent pathway acting on AtHAK5 during low-K+ signaling has been recently provided (Brauer et al., 2016). The INTEGRIN LINKED KINASE1 (ILK1) interacts with AtHAK5 and promotes its accumulation, likely by phosphorylation of AtHAK5, albeit this could not be proved *in vitro*. ILK1 activity contributes to growth during extreme K+ limitation where AtHAK5 is the only transport system contributing to K+ uptake. ILK1 interacts with the calmodulin-like protein 9 (CML9), which invokes Ca2+ signaling, and both proteins were needed to promote AtHAK5 accumulation in the membrane fraction of *Nicotiana benthamiana* leaves, leading to the suggestion that ILK1 and CML9 promote HAK5 maturation and transport from the endoplasmic reticulum to the plasma membrane. It remains to be investigated whether ILK1 functions in parallel or in coordination with the CIPK23/CBL pathway to modify AtHAK5 activity.

In addition to the interaction with kinases, the modulation of KT/HAK/KUP transporters through physical interactions among different subunits (homomers or heteromers formation) has been suggested. Physical self-interaction among subunits of AtKUP4 has been described (Daras et al., 2015). In the event that KT/ HAK/KUP proteins formed a tetrameric structure similar to that of *Shaker* channels, the conserved sequence GVVYGDLGTSPLY in the first TM fragment of each subunit would line the pore, where the three conserved glycine residues of each subunit may operate as a selectivity filter (Rodriguez-Navarro, 2000).

In summary, the integration of post-translational regulation, including a specific Ca2+ signature, protein interactions, phosphorylation events, and reallocation of transporters to the plasma membrane, as well as the transcriptional regulation governed by plasma membrane hyperpolarization, ROS production, and hormonal response pathways, all of them integrate in the regulation of HAK1-like K+ uptake systems in plant cells under different K+ starvation conditions. **Figure 3** summarizes the transcriptional and post-translational regulation of AtHAK5.

The *Arabidopsis* mutant lacking HAK5 and AKT1 still takes up K+ and shows residual growth at external K+ concentration above 1 mM, indicating the existence of additional compensatory transport system(s) (Pyo et al., 2010; Rubio et al., 2010). Because this low-affinity uptake is largely sensitive to Ca2+ and other divalent metals, permeates Cs+ , and is inhibited by cyclic nucleotides, non-selective cyclic nucleotide-gated cation channels (CNGC) are likely candidates for this alternative system (Caballero et al., 2012). Indeed, genetic data have implicated AtCNGC3 and AtCNGC10 in K+ uptake because knock-out and knockdown lines have reduced K+ contents (Kaplan et al., 2007). Moreover, AtCHX13, a plasma membrane cation/proton antiporter up-regulated by K+ starvation, has been involved in root K+ uptake (Zhao et al., 2008). AtKUP7 is preferentially expressed in *Arabidopsis* roots, and may also be instrumental in K+ uptake and in K+ loading into xylem sap, affecting K+ translocation from roots to shoots (Han et al., 2016). In rice, OsHAK1 and OsAKT1 fulfill the same functions than the *Arabidopsis* counterparts HAK5 and AKT1 (Li et al., 2014; Chen et al., 2015; Nieves-Cordones et al., 2017). In addition, OsHAK5 has been related to high-affinity K+ uptake and in the release of K+ into the xylem (Nieves-Cordones et al., 2016b). **Table 2** summarizes the range of K+ concentrations in soil at which each AKT1-like channel and HAK1-like transporter contributes to high-affinity K+ uptake in *Arabidopsis* and rice (Aleman et al., 2011; Nieves-Cordones et al., 2016a).

Of note is that HKT2-like transporters have been implicated in K+ nutrition in cereals. The rice protein OsHKT2;1 provides a major pathway for root high-affinity Na+ uptake that supports plant growth under limiting K+ supply (Horie et al., 2007; Haro et al., 2010). Under K+ starvation, Na+ can partially compensate for K+ as osmoticum and change balance (Rodriguez-Navarro and Rubio, 2006; Alvarez-Aragon and Rodriguez-Navarro, 2017), and indeed high-affinity Na+ uptake has been observed in roots of several species (Haro et al., 2010). Rates of Rb+ influx between WT and *hkt2;1* roots did not reveal significant differences, but *hkt2;1* had lower Na+ contents (Horie et al., 2007). Thus, the possible involvement of OsHKT2;1 in root K+ uptake could not be verified. Although the activity of HKTs may influence the K+ status of plants, particularly under saline stress, HKTs appear to be determinants for salt tolerance with no significant role in K+ nutrition (Hamamoto et al., 2015).

#### COMPARTMENTATION AND STORAGE INTO VACUOLES

Once K+ is inside the root symplast, it may be stored in vacuoles locally, or transported to the shoot *via* xylem and accumulated in aerial tissues.  In K+ -sufficient plants, K+ content can reach up to 10% of plant dry weight, thereby exceeding the ca. 2% that supports near-maximal growth rates (White and Karley, 2010). The vacuolar K+ pool plays a chief biophysical function, i.e. the lowering of osmotic potential to draw water, generate turgor, and drive cell expansion. Because vacuoles occupy most of the intracellular volume of plant cells and are the main cellular reservoir for K+ , changes in tissue K+ concentration are largely a reflection of the dynamics of the vacuolar pool. Cytosolic K+ concentration will decline below the optimal set point of 80–100 mM only when the vacuolar K+ reserve has been depleted below the thermodynamic equilibrium with the cytosolic pool (Walker et al., 1996). Conversely, surplus K+ is placed into the vacuole to maintain cytosolic K+ within narrow limits independently of K+ abundance in the growth medium.

In contrast to the plasma membrane, accumulation of K+ in the vacuole depends on the coordinated activity of tonoplast H+ pumps and secondary K+ transporters that link K+ fluxes to the dissipation of the pH gradient or the electrical membrane potential created by the asymmetric distribution of charges. The vacuolar H+ -ATPase (V-ATPase) and the pyrophosphatase of the tonoplast (PPase) pump H+ toward the vacuolar lumen and generate pH gradients of 1–2 pH units (acidic inside) and an electrical charge (membrane potential) of 20–40 mV that is positive in the vacuolar lumen relative to the cytosol. This means that positively charged K+ ions are excluded from K+ -replete vacuoles unless transport is coupled to an energydependent uptake mechanism, whereas efflux is driven by vacuolar channels permeating K+ downhill its electrochemical gradient. A K+ /H+ antiporter energized by the pH gradient across the tonoplast was long suggested to catalyze vacuolar K+ accumulation (Walker et al., 1996; Carden et al., 2003), but the molecular identity of the underlying transporter(s) has remained elusive until recently. Vacuolar NHX-type exchangers have been shown to serve this critical function in plant cells (Venema et al., 2002; Venema et al., 2003; Leidi et al., 2010; Bassil et al., 2011; Barragan et al., 2012).

NHX exchangers were originally described as Na+ /H+ antiporters able to confer salt tolerance by driving the sequestration of excess Na+ into vacuoles (Blumwald, 2000). However, the underlying mechanism remained uncertain because the salt tolerance of transgenics overexpressing NHX proteins from various sources did not always correlate with enhanced Na+ accumulation (Jiang et al., 2010). Moreover, biochemical studies established that NHX proteins catalyze Na+ /H+ and K+ /H+ exchange with similar affinities (Venema et al., 2002). The recent meta-analysis of a large number of publications reporting tolerance phenotypes imparted by exchangers of the Cation/Proton Antiporter Family 1 (CPA1, which includes NHX proteins) concluded that the effect on K+ status was generally more pronounced than on Na+ content (Ma et al., 2017). An informative work showed that overexpression of the AtNHX1 in tomato induced K+ -deficiency symptoms despite transgenic plants having greater K+ contents than controls (Leidi et al., 2010). The intense sequestration of K+ in NHX1-overexpressing plants reduced cytosolic K+ activity, primed the induction of the high-affinity K+ uptake system, and elicited an array of metabolic and hormonal disorders related to K+ deprivation (Leidi et al., 2010; De Luca et al., 2018). Notwithstanding these unintended effects resulting from NHX overexpression, NHX proteins do increase salt tolerance, presumably because retention of cellular K+ is a requisite for adaptation to a saline environment (Jiang et al., 2010). Salinity stress elicits depolarization of the root plasma membrane and ROS production, both of which open outward-rectifying K+ channels that discharge K+ to re-build the membrane potential (Shabala and Pottosin, 2014). The salinity-induced K+ loss implies the need to replenish the cytosolic K+ pool by withdrawing K+ stored in vacuoles (Cuin et al., 2003; Leidi et al., 2010).

Deletion of *NHX1* and *NHX2* genes encoding the two major vacuolar NHX isoforms resulted in the inability to compartmentalize K+ and, surprisingly, in sensitivity to K+ supply at concentrations that did not compromise the growth of control plants (Bassil et al., 2011; Barragan et al., 2012). Moreover, *nhx1 nhx2* mutant lines showed dysfunctional stomatal activity, with impaired opening and closure (Barragan et al., 2012; Andrés et al., 2014). The rapid uptake and release of K+ and anionic organic acids by guard cells, mostly in the vacuolar compartment, drives the movements of stomata. Changes in the volume and shape of guard cells run in parallel with intense remodeling of vacuoles (Gao et al., 2005; Tanaka et al., 2007). Disruption of K+ accumulation in the guard cells of *nhx1 nhx2* mutant plants correlated with more acidic vacuoles and the disappearance of the highly dynamic remodeling of vacuolar structure associated with stomatal movements (Andrés et al., 2014).

Electrophysiological recordings of channel activities in the tonoplast have identified fast vacuolar (FV), slow vacuolar (SV), and K+ -selective vacuolar (VK) cation channels that mediate the release of vacuolar K+ (Hedrich, 2012). The VK currents have been assigned to two-pore K+ (TPK) channels (Gobert et al., 2007). TPK1, 2, 3, and 5 of *Arabidopsis* are located in the tonoplast, while TPK4 is in plasma membrane. TPK1 currents are independent of the membrane voltage but sensitive to cytosolic Ca2+ and regulated by calcium-dependent protein kinases (CDPKs) and 14-3-3 protein binding (Latz et al., 2013). Also in *Arabidopsis*, the TPC1 channel accounts for the SV current (Peiter et al., 2005). TPC1 is voltage-dependent and non-selective, allowing K+ and Na+ to permeate toward the cytosol. Whether TPC1 also permeates Ca2+ or Ca2+ is only an effector of TPC1 gating is a matter of controversy (Hedrich et al., 2018). TPC channels are activated by a decrease in transmembrane potential and increased cytosolic Ca2+, and inhibited by low luminal pH and Ca2+. Structurally, TPC1 resembles two subunits of voltage-dependent *Shaker*-like channel fused in tandem, and two cytosolic EF hands in between (Guo et al., 2016; Kintzer and Stroud, 2016). The ubiquitous nature of TPC channels and the magnitude of the SV/TPC currents are such that TPC channels are capable of contributing substantially to cellular K+ homeostasis. Accordingly, the transcriptome of the *tpc1* loss-of-function mutant of *Arabidopsis* is reminiscent of profiles that were obtained under K+ limitation (Bonaventure et al., 2007b). However, plants lacking TPC1 function are not impaired in growth and development. This may indicate that the TPC1 channel is closed most of the time and opens upon specific inputs or under stress. Current thinking is that TPC1 is part of a Ca2+/ROS relay that propagates stress signals (Choi et al., 2014; Evans et al., 2016). The gain-of-function mutant *fou2* results in a hyperactive TPC1 channel with an altered voltage-dependent gating behavior that increases the probability of the channel to be open under physiological vacuolar potentials. As a consequence of this "leaky" channel, the *fou2* mutant plant behaves as being wounded and shows elevated levels of the stress hormone jasmonate (Bonaventure et al., 2007a).

Several KT/HAK/KUP transporters have been localized to the tonoplast (**Table 1**). They are thought to force the energetically uphill release of K+ into the cytoplasm under chronic K+ starvation, in which the cytosolic concentration of K+ could be low enough to impair the discharge of luminal K+ by tonoplast channels (Ahmad and Maathuis, 2014).

#### LONG-DISTANCE TRANSPORT AND INTER-ORGAN K+ PARTITIONING

Potassium absorbed by peripheral root cells and not compartmentalized in vacuoles must be transported to the upper parts of the plant through the xylem (Gaymard et al., 1998; Park et al., 2008; Ahmad and Maathuis, 2014). This step is critical in the long-distance distribution of K+ from roots to the upper parts of the plant, and is driven by negative pressure (pulling) created by evaporation of water from leaves. The osmotic water uptake that is caused by nutrient absorption in the root also provides a positive force, known as root pressure, from roots to xylem vessels. Under regular K+ supply, symplastic K+ diffusion to the xylem through the stele may contribute sufficiently to K+ transport from root to shoot (Yang et al., 2014). Moreover, K+ is highly mobile within plants, exhibiting cycling between roots and shoots *via* xylem and phloem (Ahmad and Maathuis, 2014). In this section, we will review K+ channels, KT/HAK/KUP carriers, and HKT transporters that are involved in long-distance transport of K+ in plants.

Potassium channels SKOR and AKT2 play an important role in K+ translocation *via* xylem and phloem. SKOR (Stelar K+ Outward Rectifier) is expressed in root stele cells (pericycle and xylem parenchyma cells) of *Arabidopsis*, where it mediates K+ secretion by xylem parenchyma cells of roots and toward the xylem vessels (Gaymard et al., 1998). SKOR, being an outward-rectifying channel, opens upon membrane depolarization to allow cytosolic K+ efflux. In addition, the gating of SKOR is sensitive to extracellular K+ concentration, with a maximum activity around 10 mM K+ . In the presence of ample external K+ , the channel opens at less negative membrane voltages, thereby minimizing the risk to serve as an undesirable K+ influx pathway. This behavior of SKOR is achieved by a complex interplay between the pore region and the "S6 gating domain" localized in the last transmembrane segment, which contains three amino acid residues, D312-M313-I314, that acquire great relevance in coupling K+ sensitivity and gating of the channel (Johansson et al., 2006). At high external K+ concentration, the pore region is more rigid and strongly interacts with the S6 gating domain stabilizing the channel in a closed state. On the other hand, with a low external K+ concentration, the pore region is less occupied, more flexible, and does not interact with the surrounding transmembrane domains anymore.

The expression of *SKOR* is inhibited by abscisic acid (ABA) synthesized during water stress. This suggests that diminished K+ transport to the xylem in response to ABA allows osmotic adjustment of roots to the drying soil (Gaymard et al., 1998). Intra- and extra-cellular acidification also induced a decrease of SKOR currents at the macroscopic and single-channel levels without affecting macroscopic gating parameters and the single channel conductance. This decrease of SKOR currents could be due to a reduction in the number of channels available for activation, which could help preventing K+ loss from roots toward the shoot tissue (Lacombe et al., 2000a). Hydrogen peroxide exhibits the opposite effect on SKOR. Treatment with H2O2 increases SKOR outward currents and decreases its half activation time. Analyses in heterologous expression systems showed that SKOR sensibility to ROS is a voltage-dependent process produced by oxidation of Cys168 located on the S3 α-helix within channel (Garcia-Mata et al., 2010). Thus, upon acute depolarization of plasma membrane induced by salinity, SKOR in xylem parenchyma cells can be rapidly activated to mediate K+ loading into the xylem. After the plasma membrane potential is restored by increased H+ -ATPase activity, SKORdependent K+ release from root stelar cells to the xylem by membrane depolarization is suppressed. Then, accumulated ROS under salinity could, in turn, activate SKOR channels to allow xylem K+ loading. This may require a highly coordinated mechanism to ensure efficient xylem K+ loading in saltstressed plants.

Large quantities of K+ recirculate from roots to shoots *via* the xylem and subsequently return to the roots *via* the phloem (Thompson and Zwieniecki, 2005; De Schepper et al., 2013). The magnitude of the K+ flux recirculated from the shoots to the roots would constitute a signal by which the growing shoots could communicate to roots their K+ requirement and regulate K+ secretion into the xylem sap (and eventually root K+ uptake). *AKT2* is mainly expressed in the phloem both in leaves and roots (Deeken et al., 2000; Lacombe et al., 2000b), where the AKT2 channel protein plays a dual role by loading K+ in source tissues and unloading K+ in sink organs (Gajdanowicz et al., 2011). AKT2 is the only weak inward-rectifier characterized in *Arabidopsis* (Dreyer et al., 2001; Cherel et al., 2014). The protein phosphatase PP2CA interacts with AKT2 to induce both the inhibition of the channel current and the enhancement of its inward rectification (Cherel et al., 2002). When expressed in mammalian COS cells and *Xenopus* oocytes, AKT2 channel exhibited two gating modes that were dependent on phosphorylation by endogenous cAMP-dependent protein kinase A (PKA) (Michard et al., 2005a,b). In mode 1, the non-phosphorylated channel behaves as a weak inward-rectifier. In mode 2, phosphorylated AKT2 is permanently open and able to conduct K+ in the inward and the outward directions. Two serine residues, S210 and S329, located in the pore inner mouth that likely undergoes conformational changes on voltagedependent movements, were identified as targets for phosphorylation (Michard et al., 2005a). Nonetheless, it was proposed that post-translational modifications in these positions alone are not enough to completely convert AKT2 from an inward-rectifying to a non-rectifying channel. A lysine within the voltage sensor enables AKT2 to sense its phosphorylation status and to change between the two modes. Replacement of the lysine by serine or arginine displays an AKT2 inwardrectifier (Michard et al., 2005a; Sandmann et al., 2011). Thus, AKT2 can modulate the membrane voltage by switching between its modes of an inward or a non-rectifying channel, respectively, and phosphorylation acts as a tool for fine-tuning (Deeken et al., 2000; Michard et al., 2005b). Depending on the cellular context, the phosphorylation status of the AKT2 channels may change, enabling them to drive either inward or outward K+ fluxes (Michard et al., 2005a). Of note is that ABA reduces *SKOR* expression in the xylem while increasing that of *AKT2*

in the phloem (Pilot et al., 2003). This dual effect reduces K+ transport to the shoots and increases delivery of K+ to the roots *via* the phloem, thus helping in maintaining a low osmotic potential in water-deprived and salt-stressed roots.