Role of Potassium-Dependent Alternative Splicing of MYB59 in the Maintenance of Potassium Concentration in Shoots of Arabidopsis thaliana

Abstract

Potassium (K) is a major plant nutrient. K⁺ is taken up by channel and transporter proteins in roots and translocated from roots to shoots via the xylem. In Arabidopsis thaliana, the K⁺ transporter NPF7.3 mediates K⁺ loading into the xylem and the transcription factor MYB59 is responsible for NPF7.3 expression. Here, we demonstrate that MYB59 is regulated by alternative splicing in response to K availability. Three splicing isoforms of MYB59 are detected in roots: an isoform with the first intron spliced out encodes a protein with the full DNA-binding motif (MYB59α), and two isoforms with the first intron retained partially or completely encode a protein missing part of the DNA-binding motif (MYB59β). Functional analysis showed that only MYB59α is capable of inducing the expression of NPF7.3. The abundance of the MYB59α isoform increased under low K, but the total abundance of MYB59 transcripts did not change, indicating that MYB59α is increased by modification of the splicing pattern in response to low K. Although MYB59α is increased by low K, NPF7.3 expression remained constant independent of K. In addition, there was no significant difference in NPF7.3 expression between an MYB59 knockout mutant and the wild type under normal K. These results suggest that an unknown mechanism is involved in NPF7.3 expression under normal K and switches roles with MYB59 under low K. We propose that the regulation of MYB59 by alternative splicing is required for the maintenance of shoot K concentration in adaptation to low K.

Introduction

Potassium (K) is an essential plant macronutrient. K⁺ is a major cation in plant cells and plays an essential role in the establishment of cell osmotic potential (Taiz and Zeiger 2006). Regulation of osmotic potential by K⁺ is involved in various physiological processes: turgor-based movements (e.g. stomatal opening and cell expansion) are facilitated by K⁺ flux in cells (Hedrich 2012), and osmotic adjustment with K⁺ is essential for adaptation to drought (Osakabe et al. 2013). K⁺ is also required as a cofactor of enzymes involved in respiration and photosynthesis (Taiz and Zeiger 2006). Plant shoots require ∼1% K (on a dry weight basis) for growth, and cytoplasmic K⁺ is maintained at 100–200 mM (Marschner 1995). The available K⁺ level in soils is of the order of parts per million, and therefore, K deficiency is a major factor limiting plant growth and crop productivity.

Plants take up K⁺ from soils via the K⁺ channel and transporter proteins in roots. In Arabidopsis thaliana, AKT1, a voltage-dependent inward-rectifying K⁺ channel (Hirsch et al. 1998), and HAK5, a high-affinity K⁺ transporter (Gierth et al. 2005, Qi et al. 2008), have been well characterized. AKT1 and HAK5 localize at the plasma membrane of root cells and permeate K⁺ into the cells. The homologous HAK transporters and the cation/H⁺ exchanger transporter CHX13 are also thought to participate in K⁺ uptake into the roots of A. thaliana (Ahn et al. 2004, Zhao et al. 2008). K⁺ is transported toward the stele via the plasmodesmata and loaded into the xylem for translocation to shoots. K⁺ xylem loading is mediated by SKOR and NPF7.3 in A. thaliana. SKOR is a voltage-dependent outward-rectifying K⁺ channel localizing at the plasma membrane of xylem parenchyma cells and effluxes K⁺ into the xylem (Gaymard et al. 1998, Johansson et al. 2006, Liu et al. 2006). NPF7.3 (also known as NRT1.5) is a K⁺/NO₃⁻ symporter and mediates xylem loading of both K⁺ and NO₃⁻ (Drechsler et al. 2015, Zheng et al. 2016, Li et al. 2017). Although SKOR and NPF7.3 share a role in K⁺ xylem loading, NPF7.3 likely contributes more under low K than SKOR (Li et al. 2017).

Expression of NPF7.3 is regulated by the transcription factor (TF) MYB59. MYB59 binds to the promoter region of NPF7.3 and induces transcription. Knockout of MYB59 reduces NPF7.3 expression, resulting in the reduction of shoot K and severe growth damage under low K (Du et al. 2019). Thus, MYB59 is required for low-K tolerance. Previously, we performed a genome-wide analysis of alterations in transcript sequences in response to a deficiency of 12 nutrients (N, K, Ca, Mg, P, S, B, Fe, Mn, Zn, Cu and Mo) in A. thaliana roots and found that the splicing pattern of MYB59 is modulated specifically by low-K treatment (Nishida et al. 2017). Under low K, the abundance of a splicing isoform of MYB59 encoding a protein with the full DNA-binding motif increased, while that of splicing isoforms encoding a truncated protein missing part of the DNA-binding motif decreased, implying that MYB59 is activated by the modulation of RNA structure. Here, we investigated the involvement of this low-K-dependent splicing regulation of MYB59 in low-K tolerance. Our data demonstrate that MYB59 is activated and plays a role in NPF7.3 expression under low-K conditions. However, the contribution of MYB59 to NPF7.3 expression was little or none under normal-K conditions, suggesting that an unknown TF plays a role in NPF7.3 expression under normal K.

Results

MYB59 splicing isoforms

We reported that three splicing isoforms of MYB59 (AT5G59780) are expressed in the roots of A. thaliana (Nishida et al. 2017). They are identical to the MYB59 transcripts registered in the Arabidopsis Information Resource (TAIR10) database (AT5G59780.1–3). MYB59 has two introns. The first is alternatively spliced (Fig. 1A). It has in-frame stop codons, and therefore, the main open-reading frame (ORF) in the isoforms retaining part or all of the first intron (isoforms #1 and #3, respectively) starts from +307 (Fig. 1A, Supplementary Fig. S1). MYB59, which is classified as an R2R3-type MYB family member, has two conserved DNA-binding domains, R2 and R3. The non-intron-retention isoform, #2, encodes a protein with the full DNA-binding domain; the intron-retention isoforms #1 and #3 encode a truncated protein missing R2 (Fig. 1B, Supplementary Fig. S1). Here, we name the ORF encoding the full protein isoform MYB59α and that encoding the truncated isoform MYB59β. In our previous study, we showed that the alternative splicing of MYB59 is regulated in response to low-K conditions in hydroponic culture (Nishida et al. 2017). We confirmed this regulation here in agar plate experiments (Fig. 1C, D). A semi-quantitative PCR (qPCR) analysis targeting the alternatively spliced region showed that the PCR product corresponding to isoform #2 increased under low K, whereas isoform #1 decreased and isoform #3 showed no clear change (Fig. 1C). These results agree with the results obtained by RNA-Seq analysis (Nishida et al. 2017). The qPCR analysis showed that the abundance of isoform #2 increased to more than seven times under low K relative to normal K (Fig. 1D).

MYB59 is activated by alternative splicing under low-K conditions

To investigate the functions of MYB59α and MYB59β, we introduced each DNA fragment into an MYB59 knockout mutant (myb59-1), which has Transfer DNA (T-DNA) insertion in the coding region of MYB59 (Supplementary Fig. S2A). Growth under low K was significantly reduced in myb59-1 relative to the corresponding wild type (Col-0) but was recovered and further improved by the introduction of MYB59α (Fig. 2A–C, Supplementary Fig. S3). The expression level of NPF7.3 under low K in myb59-1 was approximately one-fourth of that in the wild type but was significantly increased in the MYB59α-introduced lines (Fig. 2D, Supplementary Fig. S3). myb59-1 had a reduced ratio of shoot-to-root K owing to the defective root-to-shoot K translocation by NPF7.3; however, the ratio was recovered by the introduction of MYB59α (Fig. 2E–G). These results show that MYB59α encodes the active TF protein. In contrast, the MYB59β-introduced plants had a low-K tolerance comparable to that of myb59-1 (Fig. 3A–C), and NPF7.3 expression and the shoot-to-root K ratio under low K were not recovered in them (Fig. 3D–G), indicating that MYB59β encodes the inactive form. As shown earlier, isoform #2, encoding MYB59α, is upregulated, and isoforms #1 and #3, encoding MYB59β, are downregulated under low-K conditions. Thus, MYB59 is activated by the modulation of the splicing pattern in response to low K. NPF7.3 is also known as a NO₃⁻ transporter and is involved in the NO₃⁻ translocation from roots to shoots. It was confirmed that the ratio of shoot-to-root NO₃⁻ was significantly lower in myb59-1 than in the wild type under low-K conditions, and the ratio was recovered in the MYB59α-introduced lines (Fig. 4). In contrast, the shoot-to-root NO₃⁻ ratio was not recovered in the MYB59β-introduced lines.

Under normal K, the growth of MYB59α-introduced plants was significantly less than that of the wild type (Fig. 2A–C). This suggests that MYB59α inhibits the growth under normal K. NPF7.3 expression significantly increased (to 3.1 times in line #1 and to 1.9 times in line #8) and, accordingly, the shoot-to-root K ratio also increased (to 1.5 times in both lines) in the MYB59α-introduced plants relative to the wild type (Fig. 2D–G). This elevated expression of NPF7.3 in the MYB59α-introduced plants was probably caused by increased MYB59α expression (Supplementary Fig. S4C). In contrast, MYB59β expression had no effects on growth, NPF7.3 expression or K accumulation pattern under normal K (Fig. 3D–G).

The expression of SKOR, which works with NPF7.3 in K⁺ xylem loading, was not lower but rather higher in myb59-1 than in the wild type under low K (Supplementary Fig. S4). SKOR expression was lower in the MYB59α-introduced plants than in the wild type. These results show that MYB59α is not required for the expression of SKOR under low K.

The introduced fragments of MYB59α and MYB59β were fused with a sequence encoding green fluorescent protein (GFP) at the 3ʹ end (Supplementary Fig. S2). However, a clear GFP signal was not detected in any transgenic plants regardless of the K condition (Supplementary Fig. S5). In addition, our lack of detection of MYB59 in a Western blotting assay (data not shown) implies that MYB59 is unstable and does not accumulate abundantly in cells.

MYB59 is little or not required for NPF7.3 expression under normal-K conditions

Although MYB59α abundance significantly increased under low K, NPF7.3 expression did not change between normal K and low K in the wild type (Figs. 2D, 3D). This stable expression of NPF7.3 independent of K conditions was confirmed in a time-course study: we investigated the time-dependent change in patterns of MYB59 splicing and NPF7.3 expression under low K using wild-type plants grown hydroponically. The total abundance of MYB59 transcripts varied within a narrow range over the treatment period (Fig. 5A): it increased slightly to 1.8 times at day 1 of treatment but decreased gradually thereafter and was no longer different at day 7 between −K plants and +K plants. In contrast, the expression of MYB59α increased rapidly at day 1 and reached four times that before treatment at day 3 (Fig. 5B). The expression of MYB59α relative to that of total MYB59 transcripts continued to increase during treatment (Fig. 5C). However, the expression of NPF7.3 remained almost constant during treatment, and there was no significant difference at day 7 between −K plants and +K plants (Fig. 5D). This shows that NPF7.3 expression is almost unaffected by K condition.

Unlike under low K, there was no difference in NPF7.3 expression between myb59-1 and Col-0 under normal K (Figs. 2D, 3D). This indicates that MYB59α is little or not required for NPF7.3 expression in K-sufficient plants. Taken together, these results suggest that an unknown mechanism maintains NPF7.3 expression under normal K.

Discussion

Transcription of many plant genes related to low-nutrient tolerance (e.g. nutrient transporter genes) is activated in response to nutrient deficiency. This transcriptional regulation is a well-known mechanism that is essential for survival in low-nutrient conditions. Many TFs involved in the regulation of nutrient-related genes have been discovered in plants; MYB59 is one such. Attention has turned to how these TFs are functionally regulated in response to nutrient deficiency. Our data show that MYB59 itself is activated not by transcriptional regulation but by the regulation of alternative splicing in response to low K (Fig. 1D). It has been confirmed that activated MYB59 is required for NPF7.3 expression and for the maintenance of K⁺ translocation from roots to shoots under low K (Fig. 2D–G); the activation of MYB59 by alternative splicing would be essential for low-K tolerance in A. thaliana. NPF7.3 is also known to mediate NO₃⁻ xylem loading (Drechsler et al. 2015, Li et al. 2017), and our data are supporting that activation of MYB59 by alternative splicing is also required for root-to-shoot NO₃⁻ translocation under low K (Fig. 4). We found that MYB59α limits growth in K-sufficient plants (Fig. 2B, C). MYB59α expressed under normal K might cause the unnecessary activation of NPF7.3 or other downstream genes, resulting in physiological disorders and growth impairment. This indicates that MYB59 must be regulated in response to varying K conditions.

RNA splicing is facilitated by a spliceosome consisting of ‘splicing factor’ proteins. The RNA splicing of MYB59 is regulated under low K specifically (not by other nutrient deficiencies; Nishida et al. 2017), raising a possibility of a splicing factor that regulates alternative splicing of MYB59 under low K. We investigated the expression patterns of 395 splicing-related genes (http://www.plantgdb.org/SRGD/ASRG/ASRP-home.php) under low-nutrient conditions using RNA-Seq data published previously (Nishida et al. 2017) and found a putative splicing factor gene specifically induced under low K (Supplementary Fig. S6). However, as we observed no difference in the MYB59 splicing pattern between the knockout mutant of this gene and the wild type, this putative splicing factor gene is unlikely involved in the regulation of MYB59 splicing. It has been recently shown in animals that large non-coding RNA participates in RNA splicing regulation in cancer cells (Ouyang et al. 2022). We previously found large non-coding RNAs up- or downregulated under low K (Fukuda et al. 2019); the involvement of such large non-coding RNA in MYB59 splicing regulation must be considered.

The MYB59β isoform had no effect on NPF7.3 expression. However, a semi-qPCR analysis showed that MYB59β is abundantly expressed even under low K. Previous RNA-Seq analysis showed that the expression of MYB59β accounts for more than half of the total MYB59 transcripts even under low K (Nishida et al. 2017). If MYB59β has no physiological function, plants would be wasting energy on MYB59β expression. One might think that MYB59β represses NPF7.3 transcription. R3-type MYB TFs, which have only an R3 domain, repress target gene transcription in plants (Tominaga-Wada and Wada 2014). However, the MYB59β-encoded protein could not bind to the NPF7.3 promoter in an in vitro assay (Du et al. 2019). In addition, our complementation test showed that NPF7.3 expression was not decreased by the introduction of MYB59β; therefore, MYB59β unlikely affects NPF7.3 expression. Further studies are needed to understand the physiological significance, if any, of MYB59β expression.

Another important finding here is that although MYB59 was activated by low K, NPF7.3 expression remained constant independent of K. Moreover, MYB59 was little or not involved in the expression of NPF7.3 under normal K. These results suggest that an unknown TF is responsible for NPF7.3 expression under normal K. We assume that there is a ‘role switch’ between MYB59 and this unknown TF (Fig. 6). Under normal K, NPF7.3 expression probably relies on the unknown TF. When exposed to low K, the unknown TF is downregulated for unknown reasons, and instead, MYB59α is upregulated by alternative splicing and takes over the role from the unknown TF. This role switch between TFs may explain the constant NPF7.3 expression under varying K conditions. Du et al. (2019) reported that NPF7.3 transcription was significantly downregulated under low K owing to the decreased MYB59 transcription level. However, we did not observe the downregulation of NPF7.3 under low K either on the agar plate or in hydroponic culture. This difference in our results from the previous study might be due to a difference in treatment conditions: in the study by Du et al. (2019), plants were exposed to low K for 2 weeks from germination, whereas in our study, plants precultured under normal K (for 7 d in agar plate culture and for 21 d in hydroponic culture) were transferred to low-K conditions and grown for 7 d. Our results reflect an earlier response to low K than in the study by Du et al. (2019). In contrast, the MYB59 transcription level tended to be decreased by low-K treatment (Fig. 5, Supplementary Fig. S4), as Du et al. (2019) reported. However, the abundance of the active isoform MYB59α was significantly increased by alternative splicing regulation in response to low K, and the contribution of MYB59 to NPF7.3 expression accordingly increased, confirming that MYB59 is activated at least in the initial response to low K.

Our results shed new light on the involvement of alternative splicing regulation in plant nutrient responses. We previously identified more than 600 genes showing alteration in transcript sequences under low-nutrient conditions (Nishida et al. 2017). Most of these genes (>93%) showed no significant change in total transcript abundance, indicating that their responses to low nutrients had been overlooked in gene-based expression analysis. These ‘hidden’ transcriptome responses might be involved in an unknown mechanism underlying low-nutrient tolerance in plants. Very recently, Guo et al. (2022) reported that the differential RNA splicing of REGULATOR OF LEAF INCLINATION 1 (RLI1), encoding an MYB TF protein (not an ortholog of MYB59), in response to low-phosphate conditions in rice is involved in the induction of phosphate starvation signaling. Henceforth, the modulation of RNA structure should be considered as a novel gene regulation mechanism in plant nutrient responses.

Materials and Methods

Plant materials and growth conditions

Seeds of A. thaliana wild-type Col-0 and of a myb59-1 T-DNA insertion line (CS460129; Mu et al. 2009; Supplementary Fig. S2) were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA).

Plants were grown on Molecular Genetics Research Laboratory (MGRL) medium (pH 5.7; Fe-EDTA as an Fe source). Plants were precultured for 1 week on 1.2% agar medium (purified agar powder, Nacalai Tesque Inc., Kyoto, Japan) supplemented with 1% sucrose and transferred to assay agar medium supplemented with 67 µM EDTA-Na₂, 0.1% 2-morpholinoethanesulfonic acid (MES) and 3 mM KNO₃ for normal-K condition or 3 mM NaNO₃ for low-K condition. Plants treated for 7 d were used for gene expression analyses, and plants treated for 9 d were used for growth and elemental analyses. In hydroponic culture testing, plants were precultured in half-strength MGRL medium (Nishida et al. 2011) for 4 weeks and then exposed to assay medium supplemented with 33.5 µM EDTA-Na₂, 0.1% MES and 3 mM KNO₃ or NaNO₃. Plants were grown in a growth chamber at 22°C under a 16-h light/8-h dark cycle in agar plate culture or a 10-h light/14-h dark cycle in hydroponic culture.

RNA isolation and qPCR analysis

Total RNA was extracted from frozen tissues with a Plant Total RNA Mini Kit (Favorgen Biotech. Corp., Ping Tung, Taiwan), and contaminating genomic DNA was digested by DNase I (Takara Bio Inc., Shiga, Japan). cDNA was synthesized from total RNA with PrimeScript RT Master Mix (Takara Bio Inc.). qPCR used SYBR Premix Ex Taq II (Takara Bio Inc.) on a LightCycler 96 System (Roche Diagnostics K.K., Tokyo, Japan) following the manufacturer’s instructions. qPCR followed the standard curve method under the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (Bustin et al. 2009). EF1α (AT5G60390) was used as the internal control. Primers are listed in Supplementary Table S1.

Semi-qPCR

The splicing pattern of MYB59 was investigated by semi-qPCR. Primers were designed for the alternative splicing region containing the first intron of MYB59 (Fig. 1A). RNA extraction and cDNA synthesis were performed as described earlier. PCR was performed using GoTaq Master Mix on a PCR Thermal Cycler Dice Touch (Takara Bio Inc.). The thermal profile used preheating for 1 min at 95°C; 28–32 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C and a final extension for 5 min at 72°C. EF1B (AT1G57720) was used as an internal control. Primers are listed in Supplementary Table S1.

Elemental analysis

Seedlings grown on assay agar medium were separated into shoots and roots. Roots were washed with ultrapure water and blotted. Samples were weighed, dried at 70°C to determine dry weight and digested with 70% HNO₃ (120°C for 1 h) and 30% H₂O₂ (120°C for 1 h) in a heat block. Digests were diluted with 1% HNO₃ and used for the elemental analysis by inductively coupled plasma optical emission spectrometry (Agilent 5800, Agilent Technologies Japan, Ltd., Tokyo, Japan).

Nitrate measurement

Nitrate was determined following the method by Yamaguchi et al. (2016). Seedlings grown on assay agar medium were separated into shoots and roots. Tissues were washed with ultrapure water, blotted and weighted to determine fresh weight. Frozen tissues were ground and extracted in five volumes of 10 mM HCl. Cell debris was removed by centrifugation and filtration with 0.45 µm PVDF filters. The extracts were diluted 100-fold with ultrapure water and analyzed by ion chromatography (Dionex Integrion HPIC System, Thermo Fisher Scientific K.K., Tokyo, Japan) equipped with an anion exchange column (Dionex IonPac AS12A, 4ϕ × 200 mm, Thermo Fisher Scientific Inc.) and a guard column (Dionex IonPac AG12A, 4ϕ × 50 mm, Thermo Fisher Scientific Inc.). The operating parameters are as follows: injection volume, 25 µl; flow rate, 1.5 ml min⁻¹; eluent, 2.7 mM Na₂CO₃ and 0.3 mM NaHCO₃ and suppressor, Dionex AERS 500 Carbonate 4 mm (Thermo Fisher Scientific K.K.).

Plant transformation

A schematic diagram of DNA constructs is shown in Supplementary Fig. S2. The fragment of MYB59α corresponding to isoform #2 was amplified from cDNA by PCR and fused to the 3′ end of the promoter fragment of MYB59 (−2,632 to −1 bp from the start codon) by a standard overlap-extension PCR. The obtained fragment was cloned into the pCR8/GW/TOPO vector (Thermo Fisher Scientific Inc.) and sequenced. The resulting clone was subcloned into the pGWB4 plant expression vector carrying GFP tag (Nakagawa et al. 2007) by LR clonase reaction (Thermo Fisher Scientific Inc.). To construct an MYB59β-expressing plasmid, we synthesized the fragment of MYB59α with a mutation at the first start codon from ATG to ATC by PCR-based site-directed mutagenesis. The resulting clone was sequenced and subcloned into pGWB4 as described earlier. The constructs were introduced into Agrobacterium tumefaciens strain GV3101 (pMP90), and the transformants were used to transform myb59-1 by the standard floral dip method. We confirmed by reverse transcription PCR and sequencing that the transgenic lines expressed the target fragments. T₃ homozygous lines were used for experiments. Primers are listed in Supplementary Table S1.

Supplementary Data

Supplementary data are available at PCP online.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.

Funding

Japan Society for the Promotion of Science, KAKENHI Grant Numbers JP16K18667, JP18KK0426, JP22H02230 to S.N.; JP19H05637 to T.F.. Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grant Numbers JP22H04815 to S.N.; JP18H05490 to T.F..

Acknowledgments

We thank Yuriko Kobayashi (Gifu University) and the ABRC for providing Arabidopsis seeds and Momoka Saito (Hiroshima University) and Yuma Yoshida (Saga University) for their technical support.

Disclosures

The authors have no conflicts of interest to declare.

References