MaHAK5, a Potassium Transporter of Banana, Enhanced Potassium Uptake in Transgenic Arabidopsis under Low Potassium Conditions

Abstract

Potassium (K⁺) is one of the most important macronutrients for plant growth and development. It is generally accepted that the KUP/HAK/KT transporters play essential roles in K⁺ uptake at low concentrations. However, their physiological functions in bananas remain unknown. Here, we cloned MaHAK5 and analyzed its functions in banana (Musa acuminata). Gene expression analysis showed that MaHAK5 was upregulated in the roots and leaves in the early stage of low K⁺ (LK) stress. MaHAK5 was localized in the cytomembrane. The expression of MaHAK5 improved the growth of the low K⁺-sensitive yeast mutant R5421 at different K⁺ supply levels. Overexpression of MaHAK5 in Arabidopsis thaliana significantly enhanced the ability for K⁺ uptake and increased the chlorophyll content under LK stress. These results indicate that MaHAK5 plays a crucial role in maintaining K⁺ uptake in bananas.

1. Introduction

The normal growth of plants requires a number of mineral nutrients. Potassium (K⁺) is the most abundant inorganic cation and is an essential nutrient in plant cells [1]. In plants, K⁺ has significant functions related to enzyme activation, stomatal movement, osmoregulation, and maintenance of membrane potential [2,3,4]. Low availability of K⁺ in the soil causes abiotic stress to crop and strongly limits productivity [5,6]. In contrast to the stable and relatively high concentration of K⁺ (100–150 mM) in plant cytosol [7,8], K⁺ varies in the range of 0.01–1 mM in soil solutions [9]. Therefore, plants have developed a complex of transport systems to adapt to soil conditions and adjust to changing K⁺ concentrations. It has been reported that plant root cells activate different systems with suitable uptake capacities, to maintain the K⁺ supply according to the external K⁺ concentration. Classical studies on Arabidopsis thaliana and barley roots identified two major systems for K⁺ uptake: K⁺ transporter (high-affinity), and K⁺ channel protein (low-affinity) [10]. In plant genomes, these candidate genes have been confirmed as the three major transporter family members: K⁺ channels, the K⁺ uptake permeases/high-affinity K⁺ transporters/K⁺ transporters (KUP/HAK/KT), and the cation proton antiporter family.

The KUP/HAK/KT gene family is the largest potassium transporter family and is present in diverse organisms, except for Animalia [11,12]. Their ubiquity suggests that these genes play important roles in nutrient absorption and natural growth. KUP/HAK/KT genes have been identified in many species, and the gene dosage varies among species. For example, there are 13 genes belonging to the KUP/HAK/KT family in A. thaliana, 27 in Zea mays, and 56 in Triticum aestivum L. [13,14,15]. AtHAK5 from A. thaliana belongs to the KUP/HAK/KT gene family, and it was reported as a high-affinity K+ transporter participating in K⁺ uptake and translocation [16]. In rice, OsHAK5 contributes to high-affinity K⁺ uptake under potassium-deficient environments and is strongly expressed in root vascular tissues [17]. ZmHAK5 is another a high-affinity K⁺ transporter in maize. Overexpressing ZmHAK5 showed increased potassium uptake and improved growth [18]. All of these results demonstrated that HAK5 genes are related to high-affinity K⁺ uptake and that they contribute to potassium uptake under K⁺-deficient conditions.

The banana is one of the most important fruits in the world, and it is also an important staple food. The Food and Agriculture Organization (FAO) of the United Nations recognizes the banana as the fourth largest food crop behind rice, wheat, and maize. The banana is a potassium-loving plant, and its optimal output depends on the availability of potassium throughout the growth period. The quality and stress resistance of banana are also affected by potassium [19]. The yield and quality of banana were significantly improved by applying 100 kg/hm² of potassium fertilizer in plantations with depleted soil nutrients in Uganda. Bananas in low-potassium soils were more likely to be infected with Pseudocercospora, the pathogen that causes black Sigatoka on banana leaves [20]. It was also shown that the application of potash fertilizer, together with bio-organic fertilizers, could effectively improve the biological community structure of banana plantation soil and prevent the occurrence of Fusarium wilt [21]. Although much research has been conducted on the importance of potassium for bananas, no reports have characterized the genes related to K⁺ uptake.

In this study, the homolog of AtHAK5 in banana, designated as MaHAK5, was identified and functionally characterized. It was demonstrated that MaHAK5 is vital for plant growth and development in an LK environment. This study provides insights into the function of HAK genes in banana plants under LK conditions, and the findings provide a theoretical basis for the investigation of the efficient use of potassium in banana cultivation.

2. Materials and Methods

Plant materials and growth conditions: The banana cultivar (‘Cavendish’ banana (AAA) cv ‘Brazilian’ plantlets) was grown under natural light conditions. Uniform banana plantlets with 4 fully expanded leaves were transferred to a 10 L container supplied with full nutrient Hoagland solution (K⁺-sufficient) or Hoagland solution without potassium (K⁺-deficient). Each treatment had three biological replicates, and each treatment of plants was divided into roots, pseudostems, and leaves. Samples were collected at different time points. After collecting, the samples were immediately frozen in liquid nitrogen and stored at −80 °C for further RNA extraction. Nicotiana benthamiana was grown in a growth chamber at 25 °C. Arabidopsis wild-type Col-0 (WT) was cultivated under the conditions of 22 °C, 16 h light/8 h dark, with 40% humidity.

Transformation and Identification of MaHAK5 OE Arabidopsis: To generate the MaHAK5-overexpressing line (MaHAK5 OE), the MaHAK5 coding sequence was cloned into the pBI121 vector with 35S promoter and was transformed into Arabidopsis Col-0 plants. This construct was introduced into Arabidopsis using the floral-dip method [22]. Transgenic seedlings were selected on MS medium (Sigma M-5519, St. Louis, MO, USA) containing 50 μg mL⁻¹ kanamycin. Genomic DNA from kanamycin-resistant plant leaves were extracted and a PCR with gene specific primers (Supplementary Table S1) was performed for secondary screening. T1 plants positive with PCR were grown, to harvest T2 seeds. Similarly, T3 seeds from homozygous lines were harvested following the primary screening using 50 μg mL⁻¹ kanamycin, and then with PCR, to ensure stable transformation.

Extraction of genomic DNA and total RNA: The total DNA and RNA of banana plants was extracted using a Super Plant Genomic DNA Extraction Kit (Sangon, Shanghai, China) and a Plant RNA Extraction Kit (AG, Changsha, China), respectively. cDNA was synthesized using a HiScript III RT SuperMix for qPCR (+ gDNA Wiper) Reverse Transcription Kit (Vazyme, Nanjing, China) and stored at −80 °C for later use.

Gene cloning and bioinformatic analysis: The cDNA and gDNA of banana were used as templates. The PCR procedure was as follows: 95 °C for 5 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 2 min, 35 cycles; 72 °C for 5 min, and storage at 4 °C. The products were observed using a 1.0% agarose gel and purified using a DiaSpin DNA Gel Extraction Kit (Sangon, Shanghai, China). The fragments were integrated on a pClone007 Versatile Simple Vector and transformed into Escherichia coli DH5α. The positive clones were screened on LB medium including ampicillin, and sequenced by Tsingke Biotechnology Co., Ltd. (Guangzhou, China). The sequence alignment results were obtained using SnapGene. The domain of MaHAK5 was analyzed using CDD (https://www.ncbi.nlm.nih.gov/cdd accessed on 5 May 2022). Sub-cellular localization prediction was analyzed using the TMHMM Server v. 2.0 (https://www.cbs.dtu.dk/services/TMHMM accessed on 8 May 2022). ClustalX 2.1 was used for the comparison of the homology of MaHAK5 and other HAK5 proteins, and MEGA7 was used to construct a phylogenetic tree [23].

Subcellular localization of MaHAK5: The MaHAK5 sequence was cloned into the pCAMBIA1300: GFP plasmid. The plasmids harboring GFP, MaHAK5-GFP, were transferred into Agrobacterium tumefaciens (EHA105) cells, which were then introduced into leaf epidermal cells of N. benthamiana. The GFP protein was observed 2 d later using confocal microscopy (LSM 710, Carl Zeiss, Jena, Germany).

Yeast complementation assay: The sequence of MaHAK5 was cloned in the pYES2 plasmid. The plasmids (pYES2-MaHAK5 and empty pYES2 plasmid) were introduced into the R5421 yeast strain, which lacks two endogenous K⁺ transporter genes [24]. R5421 containing an empty pYES2 vector was used as a negative control. The yeast transformants were grown to OD₆₀₀ = 0.8 in yeast peptone dextrose adenine (YPDA) medium at 30 °C. Then the initial yeast cells were used to make a serial dilution (10-fold, 100-fold, and 1000-fold), and each dilution was cultured in AP medium containing difference K⁺ concentrations, as indicated. The plates were cultured at 30 °C for 3 d.

Expression profile of MaHAK5 under different K⁺ concentrations: A qRT-PCR analysis was performed to measure the expression levels of the MaHAK5 gene using a Light Cycler Real-Time system. Total RNA from all the examined tissues (root, stem, and leaf) under deficient or sufficient K⁺ was reverse transcribed to cDNA. The cDNA was diluted and amplified using Power SYBR Green PCR Master Mix (Applied Accurate Biology). Three biological replicates and three technical repetitions were performed for each sample. Then, the banana MaTUB gene was used as an internal reference gene. The sequence-related primers for qRT-PCR are listed in Supplementary Table S1. Expression levels were calculated using the 2^−ΔΔCt method.

Phenotype observation and K⁺ content analysis of transgenic Arabidopsis: WT and MaHAK5 OE seedlings were cultivated in MS medium with various K⁺ concentrations (0.01 mmol/L, 0.02 mmol/L, and 5 mmol/L) for 14 d. After rinsing with deionized water, the fresh weight of plants was measured.

For determination of chlorophyll content, the shoots of two-week-old Arabidopsis WT and transgenic plants were collected and weighed (n > 40). The chlorophyll was extracted in 80% acetone in the dark, as described by Feng et al. [25]. The chlorophyll content was calculated using MacKinney’s-specific absorption coefficients, as described in [26]. The total chlorophyll content was recorded as mg of chlorophyll per gram of fresh shoots.

For K⁺ content measurement, Arabidopsis normally grown for 30 d was then cultured in Hoagland medium containing different K⁺ concentrations (0.02 mmol/L and 5 mmol/L) for 7 d. Plants were dried at 80 °C and then crushed into powder. The samples were treated in a muffle furnace at 300 °C for 2 h and 575 °C for 12 h, then dissolved in 0.1 M HCl. The content of K⁺ was measured using an atomic emission spectrometer 4100-MP AES spectrometer (Agilent, Santa Clara, CA, USA).

3. Results

3.1. Cloning and Bioinformatic Analysis of MaHAK5

Protein sequences of AtHAK5 from Arabidopsis were used as queries to search against the candidate MaHAK5 gene using BLASTP. We cloned the MaHAK5 cDNA sequence of 2259 bp and gDNA sequence of 2933 bp from the banana cultivar ‘Brazilian’. There were seven introns in the MaHAK5 gene (Figure 1a). Sequence analysis of MaHAK5 revealed that it encoded a protein of 752 aa with a calculated molecular mass of 83.33 KD, containing 12 putative transmembrane domains, and was primarily composed of α-helix (52.66%), β-pleated sheet (11.97%), and random coil (35.37%), and with a “K_trans” domain that is specific to KUP/HAK/KT transporters (Figure 1a,b). Sequence alignment showed that MaHAK5 shared 51–98% identity with its counterparts from Arabidopsis (51.65%), wild banana (97.88%), tobacco (51.98%), rice (52.44%), soybean (55.78%), wheat (52.32%), and maize (51.52%) (Figure S1). Sequence alignments in the predicted “K trans” domain showed that MaHAK5 shared 70% similarity with OsHAK5, suggesting that MaHAK5 may have a similar function to OsHAK5.

The phylogenetic tree drawn by MEGA 7.0 showed that the HAK5s of maize, rice, and wheat were closer to MaHAK5 than those of tobacco, soybean, and Arabidopsis; a pattern that reflects the evolutionary relation between monocots and dicots (Figure 1c).

3.2. Subcellular Localization of the MaHAK5 Protein

Most of the KUP/HAK/KTs have been reported to be localized in the membranes of plant cells. To survey the sub-cellular localization of MaHAK5, we transiently expressed the recombinant gene MaHAK5-GFP in N. benthamiana leaves. As shown in Figure 2, MaHAK5-GFP fusion protein was observed in the cytomembrane, whereas GFP protein alone occurred in the nucleus and diffused in part of the cytoplasm.

3.3. MaHAK5 Functions in Potassium Uptake in Yeast

Previous reports revealed that transformation of OsHAK5 in the yeast strain R5421, which is deficient in K⁺ uptake growth, exhibited normal growth under LK conditions [17]. To investigate the function of MaHAK5 in potassium uptake, the mutant strain R5421 was transformed with pYES2-MaHAK5 and an empty control vector, and the transformants were further cultured on AP medium with various K⁺ concentrations, as indicated. As shown in Figure 3, MaHAK5 complemented the K⁺ uptake-deficient phenotype of R5421 on AP medium with various K⁺ concentrations, while the control strain grew slowly under low K⁺ conditions (under 2.5 mmol/L). These results suggested that MaHAK5 is responsible for K⁺ uptake, indicating that it is a high-affinity K⁺ transporter in vivo.

3.4. Expression Profile of MaHAK5 under Different K⁺ Concentrations

In many plants, HAK5 genes such as AtHAK5 are considered to be marker genes for studying the response to LK stress [16,18]. The expression levels of MaHAK5 in various tissues under different levels of K⁺ stress were analyzed using qRT-PCR. As shown in Figure 4, MaHAK5 was widely expressed in all examined tissues. After being transferred to a LK condition for 8 h, the MaHAK5 expression rose significantly in roots and leaves, while the transcript levels did not significantly change after 14 h. We also measured the transcript levels of MaHAK5 in pseudostems at different times, and this showed that MaHAK5 was not induced by LK stress in banana pseudostems. These results indicated that MaHAK5 may have a function in the K⁺ uptake in banana roots at the early stage of potassium-deficient conditions.

3.5. Overexpression of MaHAK5 Enhances K⁺ Uptake and Plant Growth in Arabidopsis

To describe the role of MaHAK5 in K⁺ uptake, we transformed wild-type Col-0 plants (WT) with the MaHAK5 coding sequence. After kanamycin selection, two T3 transgenic lines were obtained and confirmed as positive transformation by semiquantitative RT-PCR (Figure 5a). qRT-PCR analysis revealed that the MaHAK5 gene was highly expressed in two transgenic Arabidopsis lines (Figure 5b).

Previous studies showed that knockout of HAK5 in Arabidopsis resulted in a chlorotic phenotype and low K⁺ content under LK conditions [25]. We therefore examined the phenotypes of both transgenic Arabidopsis lines on MS medium. When grown under high K⁺ (5 mM KCl) conditions, the phenotypes of the WT were not significantly different from those of the transgenic lines. However, when grown under LK conditions (0.02 mM KCl) for 14 d, the WT plants developed leaf chlorosis and contained less chlorophyll than the MaHAK5 OE plants (Figure 5c,d). Furthermore, the fresh weight and K⁺ concentration of the transgenic Arabidopsis were significantly increased compared to those of the WT under LK conditions (0.02 mM KCl), suggesting that the MaHAK5 OE plants accumulated more K⁺ than the WT. These results demonstrated that MaHAK5 has K⁺ uptake activity and that it promotes the growth of plants under LK stress.

4. Discussion

The KUP/HAK/KT genes represent the largest family of potassium transporters and play a crucial role in environmental adaption and plant growth [11,12]. Currently, the family has been studied in various plants regarding K⁺ acquisition, salt tolerance, and K⁺ translocation [11,27]. Recently, 24 KUP/HAK/KT genes were identified in the Musa A genome [28]. However, their physiological roles remained uncharacterized in banana. In this study, we reported that MaHAK5 is a cytomembrane-located potassium transporter that mediates K⁺ uptake in banana roots. The MaHAK5 complementation assay of K⁺ uptake in the yeast revealed that MaHAK5 is a high-affinity K⁺ transporter (Figure 3). However, unlike ZmHAK5, HAK homologs in maize that mediate K⁺ uptake under extremely LK (0.01 mM) conditions in yeast complementation [18], MaHAK5 exhibited much lower K⁺ uptake activity than ZmHAK5 or AtHAK5. This suggests that MaHAK5 and other HAK5 homologs may utilize different regulatory mechanisms.

HAK5 homologs have been reported in various plant species, and most of these genes are upregulated under K⁺ starvation, indicating that this is a common strategy for plants in response to LK stress [16,18,29]. Here, we demonstrated that MaHAK5 showed a higher expression pattern in banana roots when plants were exposed to LK conditions for 8 h (Figure 4), a result that was consistent with those for OsHAK5 [17] and ZmHAK1 [18]. In addition, the AtHAK5 transcript increased with the time points during a longer starvation period of 7 d, showing a continuing induction of AtHAK5 transcripts [30]. However, we determined that the MaHAK5 transcript levels were only induced in banana roots and leaves with 8 h potassium starvation, while remaining normal for further deficient K⁺ treatment (Figure 4). These findings suggest that banana utilizes distinct mechanisms for MaHAK5 regulation compared with homologs in other plant species. It is necessary to explore the regulatory mechanism underlying MaHAK5 response to LK stress in the future.

Overexpressing the K⁺ transporter genes in Arabidopsis is a feasible way to explore their function in K⁺ uptake activity [17,18,30]. For instance, increasing the expression level of AtHAK5 enhanced root growth and K⁺ accumulation in Arabidopsis under LK conditions (Zhao et al. 2016). ZmHAK5-overexpressing maize plants showed an increased K⁺ uptake activity in their roots and improved growth. By contrast, ZmHAK1-overexpression in maize plants led to chlorosis in older leaves, due to the altered K⁺ distribution [18]. In the current study, to gain a better understanding of the function of the potassium transporter activity of MaHAK5 with LK stress, we generated MaHAK5 OE transgenic Arabidopsis. We determined that the MaHAK5 OE transgenic Arabidopsis grown on LK medium developed less chlorosis in leaves compared to those of WT seedlings (Figure 5). Notably, overexpression of MaHAK5 in Arabidopsis accumulated a higher K⁺ content than in the WT plants with LK (0.02 mM) supply, and the K⁺ content in MaHAK5 OE Arabidopsis plants was not significantly different from that in the WT at normal K⁺ (5 mM) supply. These results indicated that MaHAK5 is a high-affinity potassium transporter. Overexpressing MaHAK5 in banana may provide an efficient way to improve K⁺ utilization, especially under LK conditions.

5. Conclusions

In conclusion, our study revealed the physiological function of MaHAK5, a member of the KUP/HAK/KT family in banana. The expression levels of MaHAK5 in banana roots and leaves were increased by K⁺ deficiency at the early stage. Heterologous expression of MaHAK5 complemented the growth of a K⁺-uptake-deficient yeast mutant in LK conditions. We also determined that overexpressing MaHAK5 improved the tolerance to LK stress and increased the K⁺ content in the whole plant.