Calcium/calmodulin-dependent protein kinase OsDMI3 positively regulates saline-alkaline tolerance in rice roots

ABSTRACT

Soil saline-alkalization is a major environmental stress that impairs plant growth and crop productivity. Plant roots are the primary site for the perception of soil stresses; however, the regulation mechanism engaged in the saline-alkaline stress response in plant roots is not well understood. In this study, we identified how a rice Ca²⁺/calmodulin-dependent protein kinase, OsDMI3, confers saline-alkaline tolerance in rice root growth. We measured the OsDMI3 activity by an in-gel kinase assay, Na⁺ content by NaHCO₃ treatment, and Na⁺ and H⁺ fluxes by noninvasive micro-test technology (NMT). Furthermore, a real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed to identify the genes upregulated in response to NaHCO₃ treatment in rice roots. The results showed that NaHCO₃ significantly increased OsDMI3 expression and activity in rice roots. This was consistent with the results of Na⁺ content and NMT that indicated OsDMI3 promoted root elongation under saline-alkaline stress by reducing root Na⁺ and H⁺ influx. Moreover, real-time RT-PCR analysis revealed that OsDMI3 up-regulated the transcript levels of OsSOS1 and PM-H⁺-ATPase genes OsA3 and OsA8 in saline-alkaline stressed rice plants. Collectively, our results suggest that OsDMI3 could promote saline-alkaline tolerance in rice roots by modulating the Na⁺ and H⁺ influx. These findings provide an important genetic target for protection of growth in rice exposed to saline-alkaline stress.

Introduction

Soil salinity often co-occurs with alkalinity, causing complex damage at a high pH and a high concentration of toxic Na^+.1,2 This effect is known as soil saline-alkalization and is also a major abiotic stress reducing the growth and yield of rice, which is one of the most important salt-sensitive cereal crops .³ Plant roots are the primary site for the perception of soil stresses. Thus, elucidation of the mechanisms by which rice roots respond to saline-alkaline stress will allow for a better understanding of rice growth in saline-alkaline soils .⁴ In Arabidopsis and maize, salt overly sensitive (SOS) 3-like calcium-binding protein 3 (SCaBP3)/calcineurin B-like7 (CBL7), ⁵ 14-3-3 protein, ⁶ and maize Na⁺ content under saline-alkaline condition 1 (ZmNSA1)⁷ have been shown to promote root growth and saline-alkaline tolerance by regulating the plasma membrane Na⁺/H⁺ antiporters and PM-H⁺-ATPase activities. Another study in which various rice genotypes were screened showed that a highly efficient Fe acquisition system together with a large root system may underpin the high tolerance of rice to saline-alkaline stress .⁸ However, currently, the response mechanism of rice roots to saline-alkaline stress remains largely unknown.

Calcium and calmodulin (CaM)-dependent protein kinase (CCaMK), a plant-specific protein kinase, has been shown to be a crucial regulator of root nodule and arbuscular mycorrhizal symbioses .^9,10 In rice, CCaMK (OsDMI3) is not only required for regulating rhizobial and mycorrhizal symbioses^11,12 but is also a positive regulator of abscisic acid responses, including seed germination, root growth, antioxidant defense, and tolerance to both water stress and oxidative stress .^13–15 However, there is no evidence of OsDMI3 being involved in the response to saline-alkaline stress. In this study, we aimed to verify whether OsDMI3 is required for rice growth under saline-alkaline stress. Furthermore, we measured OsDMI3 activity, Na⁺ content, and Na⁺ and H⁺ fluxes and performed real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis to identify the mechanism by which OsDMI3 positively modulates rice root growth under saline-alkaline stress.

Materials and methods

Plant materials

Rice (Oryza sativa) plants used in this study include Nipponbare (WT), the overexpressing (OE) lines of OsDMI3 and the knockout lines (KO) of osdmi3 .¹⁵ The OsDMI3 OE mutant plants were generated by the Agrobacterium-mediated transformation method. The osdmi3 KO mutant plants were generated using the CRISPR/Cas9 system. Homozygous T3 seeds of the transgenic plants were used for further analysis. Rice seedlings were grown at 22–28°C under a 14 h light and 10 h dark photoperiod. For saline-alkaline sensitivity assays, 3-d-old seedlings with the same root length (~1 cm) were transferred to a hydroponic culture solution containing 75 mM NaHCO₃ (pH 8.0).

Kinase activity assay of OsDMI3

OsDMI3 activity was determined by an in-gel kinase assay using myelin basic protein (MBP) as a substrate, as described previously .¹⁵ Three-day-old seedlings were treated with 75 mM NaHCO₃ (pH 8) for 0, 1, 3, and 6 h. The protein (100 mg) extracted from the rice roots was immunoprecipitated with an anti-OsDMI3 antibody (2 mg, Abmart, China)¹⁶ and then incubated with 1 mg MBP (Sigma-Aldrich) in a reaction buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.5 mM CaCl₂, 2 mM CaM [Sigma-Aldrich],10 mCi [γ³²P]-ATP [3000 Ci mM⁻¹]) at 30°C for 30 min. The reaction products were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography using a storage phosphor screen (Typhoon TRIO, Amersham Biosciences).

Measurement of Na⁺ content

In order to measure the root Na⁺ contents, 3-d-old seedlings were treated with 75 mM NaHCO₃ (pH 8.0) for 24 h. The rice roots were collected and dried at 80°C for 24 h. The samples were weighed (10–15 mg) and digested in 400 μL nitric acid at 90°C for 8 h. After diluting with distilled water, the Na⁺ content was measured by inductively coupled plasma-optical emission spectrometry (Optima™ 2100 DV, Perkin Elmer, USA).

Measurement of net Na⁺ and H⁺ fluxes using noninvasive micro-test technology (NMT)

For Na⁺ and H⁺ fluxes, 3-d-old seedlings were treated with 75 mM NaHCO₃ (pH 8.0) for 24 h. The Na⁺ and H⁺ fluxes were measured at the primary root meristem zone (~500 μm form the root tip) using NMT (Younger, USA). The NMT measurement procedures were according to the method of Cao et al. .⁷

Real-time RT-PCR

Real-time RT-PCR analyzes were conducted as described previously .¹⁵ The cDNA was amplified by PCR using the following primers: OsDMI3 forward 5ʹ-TGCTCTTCGCTTGTGCTTCCAGATG-3ʹ and reverse 5ʹ-GGTCGAACACCTCGTCCAGCTT-3ʹ; OsSOS1 forward 5ʹ-ATACTGAGTGGGGTTGTTATTGC-3ʹ and reverse 5ʹ-AAAGGTAAATTTCAAAAGGTACATGG-3ʹ; OsA1 forward 5ʹ-TGGGCACATGCACATAGGA-3ʹ and reverse 5ʹ-GCTCACTGTAGCCGGTCTTCTC-3ʹ; OsA3 forward 5ʹ-AATTCTGCAATCACCTACGTGTACTT-3ʹ and reverse 5ʹ-GCTGGAGCAGGAGGGACAA-3ʹ; and OsA8 5ʹ-TGTTTAACCTACAACACGACAATGC-3ʹ and reverse 5ʹ-AATGGGATGGGAAAGGAAAATAC-3ʹ. To normalize the sample loading variance, the rice glyceraldehyde-3-phosphate dehydrogenase gene served as the internal control with primers forward 5ʹ-CTTCATAGGAATGGAAGCTGCGGGTA-3ʹ and reverse 5ʹ-CGACCACCTTGATCTTCATGCTGCTA-3ʹ. The relative expression levels of the target genes were calculated as x-fold changes relative to the wild-type with treatment (0 h). Representative results from one of the independent repeats were shown, and the data indicate mean of three technical replicates ± standard error of the mean (SEM).

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OsDMI3, LOC_Os05g41090; glyceraldehyde-3-phosphate dehydrogenase, LOC_Os02g38920; OsSOS1, LOC_Os12g44360; OsA1, LOC_Os03g48310; OsA3, LOC_Os12g44150; OsA8, LOC_Os03g01120.

Results

NaHCO₃ induces the expression and activity of OsDMI3 in rice roots

Since OsDMI3 is highly expressed in the root and leaf, ¹¹ the expression of OsDMI3 in the shoots and roots were examined with 75 mM NaHCO₃ treatment. The real-time RT-PCR results showed that OsDMI3 was induced by NaHCO₃ in the roots compared with that in the shoots (Figure 1a). To further investigate the effects of NaHCO₃ on the activity of OsDMI3 in the roots, we performed an in-gel kinase assay with MBP as a substrate. Treatment with 75 mM NaHCO₃ induced an increase in the activity of OsDMI3 (Figure 1b) in rice roots. A significant increase in the activity of OsDMI3 was observed 1 h after NaHCO₃ treatment (Figure 1b). These results suggest that OsDMI3 may be involved in the response of rice roots to saline-alkaline stress.

Figure 1.

NaHCO₃ induces the expression of OsDMI3 and the activity of OsDMI3 in rice roots

(a) Relative transcript levels of OsDMI3 by real-time reverse-transcription polymerase chain reaction (RT-PCR) in the roots and shoots of rice seedlings treated with 75 mM NaHCO₃. Values are means ± standard error of the mean (SEM) of three independent experiments. (b) The activity of OsDMI3 in the roots of rice seedlings treated with 75 mM NaHCO₃. The activity of OsDMI3 was analyzed by an in-gel kinase assay assay using myelin basic protein (MBP) as a substrate (top). Corresponding Coomassie staining is also indicated (bottom). Experiments were repeated at least three times with similar results.

OsDMI3 is required for root growth under saline-alkaline stress

To verify the role of OsDMI3 in saline-alkaline tolerance in rice roots, 3-d-old seedlings of wild-type, OsDMI3 OE, and osdmi3 KO¹⁵ were treated with 75 mM NaHCO₃. Without treatment, no difference in growth was observed between the wild-type and mutants (Figure 2a-c). After 3 d of NaHCO₃ treatment, OsDMI3 OE exhibited an increase in root length and fresh weight compared with that in the wild-type, whereas osdmi3 KO exhibited the opposite phenotype (Figure 2a,d,e). These observations suggest that OsDMI3 confers saline-alkaline tolerance in root growth.

Figure 2.

OsDMI3 is required for root growth under saline-alkaline stress

(a) Analysis of the NaHCO₃-response phenotype in the roots of wild-type, OsDMI3 overexpressing (OE), and osdmi3 knockout (KO) seedlings. Three-day-old seedlings were transferred to culture solution without or with 75 mM NaHCO₃. Photographs were taken 3 d after transfer for control seedlings and NaHCO₃ treatment seedlings. Scale bar, 1 cm. (b) and (d) Analysis of root length of seedlings in (a). (c) and (e) Analysis of fresh weight of seedlings in (a). Approximately 50 seeds of each transgenic line were analyzed per replicate for each treatment in (b) to (e). In (b) to (e), experiments were repeated at least three times with similar results. Values are means ± standard error of the mean (SEM) of three independent experiments. Means denoted by the same letter did not significantly differ at P < .05 according to Duncan’s multiple range tests.

OsDMI3 regulates root Na⁺ and H⁺ fluxes under saline-alkaline stress

Saline-alkaline stress reflects the combined effect of Na⁺ toxicity and damage caused by high pH levels. To identify the mechanism by which OsDMI3 regulated root growth under saline-alkaline stress, whether by modulating Na⁺ content or affecting the H⁺ flux, we determined the root Na⁺ content in wild-type, OsDMI3 OE, and osdmi3 KO treated with 75 mM NaHCO₃. A lower accumulation of Na⁺ was noted in OsDMI3 OE roots and a higher accumulation of Na⁺ was noted in osdmi3 KO roots than that in wild-type roots (Figure 3a). We also measured Na⁺ flux at the root meristem zone of seedlings by using NMT. After NaHCO₃ treatment for 1 d, wild-type roots exhibited a pronounced Na⁺ influx. Furthermore, OsDMI3 OE showed a low Na⁺ influx, but osdmi3 KO showed a high Na⁺ influx (Figure 3b). These results confirmed that OsDMI3 is essential for the regulation of root Na⁺ flux under saline-alkaline stress. Next, we measured the H⁺ flux at the root meristem zone of wild-type and mutants. Similar to the results for the Na⁺ flux, H⁺ influx was significantly low in OsDMI3 OE but high in osdmi3 KO (Figure 3c) roots, indicating that OsDMI3 also regulates root H⁺ flux under saline-alkaline conditions.

Figure 3.

OsDMI3 regulates root Na⁺ and H⁺ fluxes in the root under saline-alkaline stress

Na⁺ content (a), Na⁺ flux (b), and H⁺ flux (c) in the roots of wild-type, OsDMI3 overexpressing (OE), and osdmi3 knockout (KO) seedlings with or without NaHCO₃ treatment. Three-day-old seedlings were treated with 75 mM NaHCO₃ for 24 h. The Na⁺ and H⁺ fluxes were measured using noninvasive micro-test technology (NMT). Data are means ± standard error of the mean (SEM) of six independent experiments. Means denoted by the same letter did not significantly differ at P < .05 according to Duncan’s multiple range tests.

OsDMI3 mediates transcriptional regulation of OsSOS1 and PM-H⁺-ATPase genes under saline-alkaline stress

In plants, Na⁺/H⁺ antiporter is an essential Na⁺ transporter conferring a cellular Na⁺ flux, ¹⁶ and PM-H⁺-ATPases is the major pump mediating root H⁺ efflux .⁶ To further investigate the mechanism by which OsDMI3 regulates the Na⁺ and H⁺ fluxes under saline-alkaline stress, transcription levels of Na⁺/H⁺ antiporter gene (OsSOS1) and several NaHCO₃-induced PM-H⁺-ATPase genes (OsA1, OsA3, OsA8) were analyzed .^8,17 Our results showed that NaHCO₃ induced increased transcription levels of these genes in the roots of wild-type rice; the increases in the transcription levels of OsSOS1, OsA3 and OsA8 were further enhanced in OsDMI3 OE roots but inhibited in osdmi3 KO roots (Figure 4). These results suggest that OsDMI3 mediates the transcriptional upregulation of OsSOS1, OsA3, and OsA8 under saline-alkaline conditions.

Figure 4.

OsDMI3 mediates transcriptional regulation of OsSOS1, OsA3, and OsA8 under saline-alkaline stress

The transcript levels of OsSOS1, OsA1, OsA3, and OsA8 in the rice roots (genotypes as indicated) with 75 mM NaHCO₃ for 6 h. Total RNA was isolated from various samples and subjected to real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis. Data are means ± standard error of the mean (SEM) of three independent experiments.

Discussion

Saline-alkaline soils are characterized by both high salinity and high alkalinity due to the hydrolysis of two sodium carbonates (NaHCO₃ and Na₂CO₃) .¹⁸ In general, root tips of higher plants display high sensitivity to environmental stimuli; therefore, maintaining the root cell pH and Na⁺ homeostasis is necessary for the plant saline-alkaline stress response. In this study, we identified that OsDMI3 is required for the response to saline-alkaline stress in rice roots. Treatment with NaHCO₃ markedly induced the expression and activity of OsDMI3 in rice roots. Under NaHCO₃ treatment, the OsDMI3 OE exhibited higher root length, fresh weight, and lower Na⁺ and H⁺ influx than wild-type. By contrast, osdmi3 KO showed reduced tolerance to saline-alkaline stress.

The root Na⁺ homeostasis is substantially mediated by SOS1 Na⁺/H⁺ antiporter transporter .¹⁶ In addition to a reduced Na⁺ influx, OsDMI3 conferred an upregulation of OsSOS1 expression with NaHCO₃ treatment, indicating that OsDMI3-mediated Na⁺ flux is ascribed to its regulatory role of the transcription of OsSOS1 in rice roots. This showed that increased PM-H⁺-ATPase activity is crucial for mediating the root H⁺ efflux, which activates SOS1 Na⁺/H⁺ antiporter and promotes saline-alkaline tolerance .^19–21 Moreover, we showed that OsDMI3 can modulate the transcript levels of NaHCO₃-induced PM-H⁺-ATPase genes, including OsA3 and OsA8. These findings suggest that OsDMI3-mediated regulation of Na⁺ and H⁺ influx is ascribed to the transcription of OsSOS1 and PM-H⁺-ATPase genes.

It is well-known that the calcium signal-activated SOS pathway plays a critical role in plant Na⁺ homeostasis under salt stress .^22,23 SOS2, a calcium-binding protein, can phosphorylate SOS1 to promote salt tolerance by driving Na⁺ transportation .²⁴ The PM-H⁺-ATPases activity can be regulated by the phosphorylation statue of special residues with various interacting proteins .^5,6,25,26 As OsDMI3 is a protein kinase, besides its regulatory role in the transcription of OsSOS1 and PM-H⁺-ATPases, whether OsDMI3 directly regulates the transphosphorylation of these proteins to modulating the Na⁺ and H⁺ fluxes remains to be determined. More detailed investigations of the mechanism by which OsDMI3 participates in the response to saline-alkaline stress need to be performed in the future.

In summary, our results indicate that OsDMI3 functions in root growth in response to saline-alkaline stress by reducing root Na⁺ and H⁺ influx. The significantly improved root elongation under saline-alkaline stress of OsDMI3 OE indicates that OsDMI3 is a promising candidate gene for breeding saline-alkaline tolerant rice varieties.

Funding Statement

This study was supported by the National Natural Science Foundation of China [grant no. 31671606, 31971824], State Key Laboratory for Conservation, and Utilization of Subtropical Agro-bioresources [SKLCUSA-b201819].

Disclosure of potential conflicts of interest

There are no conflicts of interest to declare.