Potassium ion (K⁺) is an essential nutrient for various plant physiological functions, such as maintaining intracellular osmolality, cell turgor, and pH homeostasis. Although K⁺ is abundant on earth, the concentrations of K⁺ on the root surface is often lower than that in soil solution. Thus, plants depend on various K⁺ transport systems that mediate K⁺ uptake and transport to different plant tissues (Gierth et al., 2005; Ashley et al., 2006; Amrutha et al., 2007; Very et al., 2014). K⁺ transporters in plants can be classified into four major families: Trk/HKT, KT/HAK/KUP (KUP), CHX (cation/hydrogen exchanger), and KEA (K⁺ efflux antiporter) (Gupta et al., 2008). The KT/HAK/KUP family is the largest K⁺ transporter family and is responsible for K⁺ transport across membranes in bacteria, fungi, and plants (Li et al., 2017). The plant KT/KUP/HAK transporters were first isolated from barley (HAK1) and Arabidopsis (KUP1/KT1 and KUP2/KT2) according to their homologs to fungal HAK and bacterial KUP. Thus, the composite name of KT/HAK/KUP is used to widely represent the entire family in plants (Very et al., 2014). Mutation analysis has revealed that the 8th transmembrane domain and the C-terminus of KT/HAK/KUP play crucial roles in determining K⁺ transport capacity (Mangano et al., 2008). Moreover, plant KT/HAK/KUPs have different K⁺ affinity and are involved in cation influx and efflux (Nieves-Cordones et al., 2016). These KT/HAK/KUPs contain 10–15 transmembrane domains with both N- and C-termini at the intracellular side of the membrane, the latter being much longer (Gierth and Maser, 2007; Nieves-Cordones et al., 2016). Molecular evolution analysis has indicated that segmental duplications occurred 35.89–62.77 million years ago, resulting in its expansion in tomato (Hyun et al., 2014). Phylogenetic analysis has grouped plant KT/HAK/KUPs into four clusters (Rubio et al., 2000; Banuelos et al., 2002). Most of the KUPs in cluster I function in high-affinity K⁺ uptake, whereas cluster II are involved in low-affinity K⁺ transport (Gupta et al., 2008). To date, 13 and 27 KT/HAK/KUP gene family members have been identified from Arabidopsis and rice, respectively (Rubio et al., 2000; Maser et al., 2001; Banuelos et al., 2002; Gupta et al., 2008). Expression analysis suggests that most members of the Arabidopsis KT/HAK/KUP family expressed in the roots, siliques, leaves, and flowers. AtHAK5 expression is induced under conditions of K⁺ deprivation. Ten AtKT/KUPs expressed in root hairs and five of them expressed in root tip cells, thereby implying their role in K⁺ uptake (Ahn et al., 2004). In rice, transcripts of 26 OsHAK genes were detected in at least 1 of the 27 tested tissues, and five genes were observed to be expressed in all tissues in all three genotypes (Gupta et al., 2008). Moreover, the expression of KT/HAK/KUP genes in other plant species also supported their possible role in K⁺-mediated multiple biological processes, such as tissue development and abiotic stress responses (Su et al., 2002; Grabov, 2007; Song et al., 2015).

Some KT/HAK/KUP genes are essential for plant growth and development (Ahn et al., 2004). Knocking out AtKT3/KUP4 results in tiny root hairs, suggesting its function in cell expansion (Rigas et al., 2001). A mutation in AtKT/KUP2 (shy3-1) induces a dwarf phenotype, which results from a reduction in cell size (Elumalai et al., 2002). ARF2 directly bind to the promoter of HAK5, regulating root hair elongation (Zhao et al., 2016). Rice phloem has relatively high OsHAK5 transcript levels that regulates K⁺/Na⁺ ratio during shoot growth (Yang et al., 2014). OsHAK1 transcript abundance is elevated in the roots of K⁺-starved rice and OsHAK1 mutants exhibit a reduction in root and shoot growth (Chen et al., 2015). The GhKT1 was found to be associated with the expansion of cotton fibers in turgor-dependent growth (Ruan et al., 2001). VvKUP1 and VvKUP2 play a role in K⁺-mediated cell expansion in grape (Davies et al., 2006). These findings highlight the importance of KT/HAK/KUP transporters in plant development and K⁺ uptake.

Members of KT/HAK/KUP family also participate in stress-related responses. OsHAK1 expression is induced by K⁺ deficiency or salt stress and it confers salt tolerance by regulating K⁺ uptake and K⁺/Na⁺ ratio (Chen et al., 2015). Knocking out OsHAK21 decreases the K⁺/Na⁺ ratio and salt tolerance (Shen et al., 2015). Constitutive overexpression of OsHAK5 in tobacco improves K⁺ accumulation during salt stress and confers increased salt resistance (Horie et al., 2011; Yang et al., 2014). KUP2/6/8 plays a positive role in drought stress response by regulating osmotic homeostasis and the abscisic acid (ABA) response in Arabidopsis (Osakabe et al., 2013). Recently, INTEGRIN-LINKED KINASE1 (ILK1) was found to interact with and promote HAK5 accumulation and positively regulate osmotic stress tolerance in Arabidopsis (Brauer et al., 2016). Together, these studies reveal the crucial role of KT/HAK/KUPs in K⁺-mediated abiotic stress response.

Cassava (Manihot esculenta Crantz) is considered as a food crop and potential biofuel crop because of its high starch production (Zidenga et al., 2012). Cassava is highly resistant to abiotic stresses, such as drought and low nitrogen (Xu et al., 2013). Abiotic stress resistance in other crops may be improved using gene resources from cassava. However, the mechanism underlying cassava resistance to abiotic stress remains less known. Advancements in sequencing technologies have facilitated gene identification and expression analysis. We previously sequenced the genomes of different cassava subspecies (including wild ancestor species and modern cultivated species; Wang et al., 2014), which allows subsequent analysis of whole gene families in cassava.

To date, members of the KT/HAK/KUP family have been well-characterized in Arabidopsis, rice, peach, tomato, Physcomitrella patens, Selaginella moellendorffii and poplar by genome-wide analyses (Rubio et al., 2000; Maser et al., 2001; Banuelos et al., 2002; Gupta et al., 2008; Gomez-Porras et al., 2012; He et al., 2012; Song et al., 2015; Nieves-Cordones et al., 2016). In the present study, we identified 21 KUP genes (MeKUPs) from the cassava genome and analyzed their phylogenetic relationship, gene structure, conserved domain, and expression profiles in response to drought, salt, osmotic, cold, H₂O₂, and ABA treatments. Our analyses reveal the transcriptional control of MeKUP genes in different genotypes and candidate KUP genes that may be potentially utilized in improving crop resistance to abiotic stress.

KUP genes play important roles in plant growth, development, and response to abiotic stresses (Osakabe et al., 2013; Zhao et al., 2016). Currently, our understanding of the role of KUP family in the drought-resistant crop cassava is limited. Here, we identified 21 KUPs from the cassava genome, which was classified into four clusters based on their evolutionary relationships (Figure 1). This is consistent with the previous classification of KUPs in Arabidopsis and rice (Rubio et al., 2000; Maser et al., 2001; Banuelos et al., 2002; Gupta et al., 2008). Moreover, the classification of cassava KUPs was further supported by conserved motif and gene structure analyses with each subgroup sharing similar motifs and exon-intron structures (Figures 2, 3). Conserved motif analysis suggested that all the identified KUPs had at least four typical motifs of K⁺ potassium transporters (Figure 2). In rice, all the 27 KUP members, except for OsHAK22, showed three conserved motifs (Gupta et al., 2008). Besides, some KUP proteins harbored a truncated K⁺ potassium transporter motif (He et al., 2012). This evidence indicates protein structure divergence among KUP family members over the course of evolution. Gene structure analysis showed that most of the MeKUP genes contained 6-10 exons, and the last exon exhibited the maximum length, which coincides with the exon-intron structure of KUP genes from Arabidopsis and poplar (Ahn et al., 2004; He et al., 2012). This indicates the conservation of the gene structure of the KUP family.

Organ expression profile analyses revealed 15 differentially expressed MeKUP genes in the stems, leaves, and storage roots of three cassava genotypes, including five (MeKUP3,-4,-8,-12, and -14) that showed high expression levels in all organs of the three cassava genotypes (Figure 4). In Arabidopsis, differentially expressed KUP genes were detected in the roots, older leaves, younger leaves, developing siliques, and flowers (Ahn et al., 2004). In rice, five KUP genes (OsHAK2, OsHAK10, OsHAK15, OsHAK23, and OsHAK25) expressed in all tissues of three genotypes (Gupta et al., 2008). In peach, KT/HAK/KUP genes expressed in nine tested tissues (Song et al., 2015). Additionally, three MeKUP genes (MeKUP1,-19, and -20) showed high expression only in the leaves of W14 (Figure 4). This phenomenon has also been observed in Arabidopsis and rice, with OsHAK5, OsHAK16, OsHAK25, AtKUP6, AKUP8, and AtKUP9 showing high expression in the leaves. Besides, the fruits and leaves of peach showed the highest number of expressed KT/HAK/KUP genes (Song et al., 2015). K⁺ is an essential nutrient for various physiological processes, particularly plant growth, development, and responses to abiotic stress (Ruan et al., 2001; Ahn et al., 2004; Osakabe et al., 2013). The high expression of KUP genes in leaves suggests its involvement in K⁺ transport during leaf development or environment adaptation.

Previous studies have revealed the fundamental role of osmotic adjustment in plant response to drought stress. Cellular osmotic balance is affected by various substances, including amino acids, sugars, and K⁺ (Osakabe et al., 2013). K⁺ uptake and efflux involve various types of channels and transporters that regulate water potential and turgor during osmotic adjustment (Very and Sentenac, 2003). Biochemical and genetic studies further support that the KUP/HAK/KT family transporters, including AtKUP2/6/8 and OsHAK1/5/21, positively regulate drought and osmotic resistances by influencing the K⁺-mediated ABA response, stomatal behavior, and osmotic homeostasis (Gierth and Maser, 2007; Grabov, 2007; Horie et al., 2011; Osakabe et al., 2013; Yang et al., 2014; Chen et al., 2015; Shen et al., 2015; Brauer et al., 2016). In the present study, we observed that drought stress induced the upregulation of several cassava KUP genes in the roots and leaves of different genotypes, suggesting their possible roles drought stress response. Generally, MeKUP genes showed similar expression profiles in Arg7 and W14, which differed from that in SC124 after drought treatment. The number of MeKUP genes upregulated by drought was significantly higher in the roots than that in the leaves of Arg7 and W14, whereas fewer genes were upregulated in the roots than in the leaves of SC124 (Figure 5). Previous studies have demonstrated that KUP genes positively regulate ABA response during lateral root formation and K⁺ efflux-mediated stomatal closure in leaves, thereby increasing plant resistance to drought and osmotic stresses (Osakabe et al., 2013). Based on this evidence, the expression diversity of KUP genes under drought stress implies differences in its roles in drought response in various cassava genotypes. For the Arg7 and W14 genotypes, a higher number of MeKUP genes are involved in drought-induced ABA responses in roots, whereas in SC124, more MeKUP genes participate in K⁺ efflux-mediated stomatal closure in the leaves under drought stress. Both functions contribute to cassava resistance to drought stress.

Additionally, the total number of strongly upregulated MeKUP genes (Log₂ based fold change >2) was higher in W14 than in Arg7 and SC124 (Figure 5). W14 has greater drought resistance than Arg7 and SC124 (Hu et al., 2016). Previous studies have demonstrated that KUP genes play a positive role in drought or osmotic stress response by affecting ABA response, stomatal behavior, and osmotic homeostasis (Gierth and Maser, 2007; Grabov, 2007; Osakabe et al., 2013). These drought or osmotic responses involve KUP-mediated K⁺ uptake from the roots, transport from vascular tissues, and efflux from the leaves (Li et al., 2017). Thus, a higher number of strongly upregulated MeKUP genes in W14 may contribute to its drought resistance.

Previous studies have demonstrated the positive role of KUP genes in osmotic, drought, salt, or ABA responses, such as, AtKUP2, AtKUP6, and AtKUP8 in Arabidopsis, and OsHAK1, OsHAK5, and OsHAK21 in rice (Horie et al., 2011; Osakabe et al., 2013; Chen et al., 2015). Transcriptome analysis has identified several MeKUP genes that were responsive to drought stress (Figure 5). Thus, there is a need to investigate the expression patterns of MeKUP genes under various abiotic stress and stress-related signal molecule treatments. In the present study, we observed that all the tested genes (MeKUP-2,-3,-4,-6,-8, and -17) showed induction after salt and osmotic treatments at several time points, which coincides with the expression patterns of AtKUP2/6/8 in Arabidopsis (Osakabe et al., 2013). Notably, MeKUP2/6/8 is the homologs of AtKUP2/6/8 according to the evolutionary analysis, thereby suggesting that it may also be involved in abiotic stress response. All tested cassava genes were induced after cold and H₂O₂ treatments, which supplies a clue for further investigation of KUP-mediated cold and H₂O₂ responses. MeKUP6 and MeKUP17 were upregulated after all the treatments, indicating that these genes may play a role in multiple stress signaling pathways (Figure 6). Based on the importance of K⁺-mediated osmotic adjustment in plant response to abiotic stress, the observed response of MeKUP genes under abiotic stress, and the nature of cassava resistance to drought, it may be important to functionally characterize these genes.

In conclusion, this study identified 21 KUPs in cassava and investigated their classification, protein motifs, and gene structure. Transcriptome analysis revealed the potential role of MeKUP genes against drought stress in different cassava genotypes. Additionally, we identified several MeKUP genes that may be utilized as candidates for improving crop resistances to multiple stresses. Further studies are required to reveal the molecular mechanisms of MeKUPs in response to abiotic stress at translational and post-translational levels by biochemical and genetic approaches.

SZ, KL, and WH: conceived the study; WO, XM, CH, WT, YY, ZD, CW, ZX, and WW: performed the experiments and carried out the analysis; WH, WO, XM, and CH: designed the experiments and wrote the manuscript.