MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples

ABSTRACT

Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples.

The changes of vacuolar pH influence many different physiological processes including plant defense responses, cell expansion, protein trafficking as well as ion homeostasis.^1-3 There is an increasing evidence that changes of vacuolar pH modulate Ca²⁺ signaling in plants as well.⁴ Meanwhile, vacuolar pH shifts the anthocyanin absorption spectrum to change the hue of tissues and organs.⁵ In the freshy fruit cells of immature fruits, the vacuolar pH is often below pH 3, but gradually increases during fruit maturation.⁶ The low vacuolar pH of fruits is the result of 2 processes: 1) transporting protons into the vacuole, which directly leads to a decrease of pH, and 2) synthesis and accumulation of organic acids within the vacuole to serve as a buffer to maintain a low vacuolar pH.⁶

On the tonoplast, 2 distinct proton pumps, vacuolar H⁺-ATPases (V-ATPases) and H⁺-pyrophosphatases (V-PPases), are responsible for transporting protons from the cytosol into the vacuole by cleaving energy-rich phosphate bonds of ATP and pyrophosphate, respectively.⁶ This process generates the electrochemical potential across the vacuolar membrane, which serves as the driving force for energy-dependent secondary transport.⁷ Among these V-ATPases and V-PPases genes, an apple V-ATPase subunit gene MdVHA-B1 has been well characterized that functions in response to and defending against salt and drought stress.^8,9 In addition to its involvement in abotic stress, MdVHA-B1 has also been identified as a key regulator of malate transport into the vacuolar and vacuolar acidification.¹⁰

The signal transduction for MdVHA-B1 involving in malate transport and vacuolar acidification has been well investigated. On one hand, MdVHA-B1 can be positively regulated by a R2R3 MYB transcription factor (TF) MdMYB1 at the transcription level;¹⁰ On the other hand, MdVHA-B1 interacts with and is phosphorylated by MdSOS2L1, an apple SOS2 (Salt-overly-sensitive 2)-like protein kinase, at the posttranslational level.¹¹ However, little is known about the relationship between MdMYB1 and MdVHA-B1.

It was reported that both MdSOS2L1 and MdVHA-B1 were induced by salt stress in our previous studies.^9,12 Interestingly, it was found that the expression pattern in response to salinity of MdSOS2L1 was highly similar to that of MdVHA-B1 (Fig. 1A). To examine their genetic interactions, the full-length sense ORFs and antisense specific cDNA fragments of MdSOS2L1 and MdVHA-B1, respectively, were genetically transformed into the apple calli. Then four types of transgenic apple calli lines were obtained and used for expression analysis: 35S::MdSOS2L1 (35S-driven MdSOS2L1 overexpression), 35S::antiMdSOS2L1 (35S-driven MdSOS2L1 suppression), 35S::MdVHA-B1 (35S-driven MdVHA-B1 overexpression) and 35S::antiMdVHA-B1 (35S-driven MdVHA-B1 suppression). PCR assays demonstrated that MdSOS2L1 positively regulated the expression of MdVHA-B1, but MdVHA-B1 did not influence the expression of MdSOS2L1. Meanwhile, MdSOS2L1 seemed not to regulate the expressions of MdVHA-B2 and MdVHA-B3, and only slightly influenced the expression of MdVHA-A (Fig. 1B). Furthermore, an anti-MdVHAB antibody specific to MdVHA-B1 and MdVHA-B3 proteins¹¹ was used for immunoblotting assays. The abundance of MdVHA-B1 positively varied with the level of MdSOS2L1 protein, while MdVHA-B3 was much lower in abundance than MdVHA-B1 in the WT calli and remained at a similar level in both WT and transgenic calli lines (Fig. 1C). Therefore, MdSOS2L1 specifically acts upstream of MdVHA-B1 to regulate its transcript level, and subsequently alters protein abundance in apple.

Figure 1.

MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples. (A) qRT-PCR analysis of MdSOS2L1, MdVHA-A and MdVHA-B1 genes in in vitro shoot cultures of wild-type ‘Gala’ apple treated with 100 mM NaCl for different times, as indicated. Each data point represents the mean ± SD of 3 independent replicates. (B) The relative expression levels of MdSOS2L1, MdVHA-A, MdVHA-B1, MdVHA-B2 and MdVHA-B3 in WT, 35S::MdSOS2L1 (35S-driven MdSOS2L1 overexpression), 35S::antiMdSOS2L1 (35S-driven MdSOS2L1 suppression), 35S::MdVHA-B1 (35S-driven MdVHA-B1 overexpression) and 35S::antiMdVHA-B1 (35S-driven MdVHA-B1 suppression) transgenic apple calli with qRT-PCR. Data is shown as mean±SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t test in different apple calli lines. n.s., P > 0.01; *P < 0.01; **P < 0.001. (C) The protein levels of MdVHA-B1 and MdVHA-B3 in WT and MdSOS2L1 transgenic apple calli. Immunoblot assays were conducted with an anti-MdVHA-B antibody, and the ACTIN protein serves as a loading control. (D) Yeast 2-hybrid assays identified the interactions between MdSOS2L1 and MdMYB1 protein. Yeast grew on SC medium minus tryptophan and leucine to select for both the bait and prey proteins (upper). (Middle) SC medium minus tryptophan, leucine, histidine, and Adenine to allow the growth of only positively interacting clones. (Lower) SC medium minus tryptophan, leucine, histidine, and Adenine plus 100ul β-gal to allow the growth of only positively interacting clones. (E) Co-immunoprecipitation assay of MdSOS2L1 with MdMYB1 in 35S::GFP and 35S::MdSOS2L1-GFP transgenic apple calli. GFP and MdSOS2L1-GFP proteins were immunoprecipitated using an anti-GFP antibody, and immunoblotted using anti-MdMYB1 or anti-GFP antibody. (F) Co-localization analysis of MdSOS2L1-RFP and MdMYB1-GFP in vivo. The full-length of MdSOS2L1 was fused to the red fluorescent protein (RFP) tag. The full-length of MdMYB1 was fused to the green fluorescent protein (GFP) tag. For each image, 2 constructs, as indicated, were transferred into protoplasts of apple calli cells and then analyzed using confocal microscopy. Yellow colors in the merged images indicate the co-localization of the 2 signals. Bars = 50 μm. (G) A model of MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples.

Since MdSOS2L1, a CBL-interacting protein kinase, acts upstream of MdVHA-B1 to regulate its expression, resulting in vacuolar acidification in apple (Fig. 1A, B).¹⁰ In addition, the Arabidopsis double mutant cbl2 cbl3 shows reduced vacuolar ATPase activity and enhanced vacuolar pH.¹ Therefore, it seems that the Ca²⁺-dependent CBL-CIPK complex is involved in the transcriptional regulation of vacuolar proton pumps. In many fruits, Ca²⁺ and K⁺ are crucial for flesh acidity and vacuolar pH. MdSOS2L1 expression was induced by CaCl₂,¹² indicating that Ca²⁺ at least partially regulated flesh acidity by modulating MdSOS2L1. In grapevines, the CBL-CIPK complex activates the K⁺ channel VvK1.2 to transport K⁺ into the cytoplasm, which is negatively correlated with fruit acidity.¹³ In addition, calmodulin (CaM) interacts with a MYB transcription factor (TF), MYB2, in Arabidopsis.¹⁴ While MYB TF PH4 plays a crucial role in vacuolar pH, which is important for flower color in petunias.¹⁵ Moreover, MdMYB1 TF transcriptionally regulates MdVHA-B1 to control vacuolar pH in apple.¹⁰ Therefore, it seems that MdMYB1 TF regulates the expression of MdVHA-B1 in response to MdSOS2L1- or CaM-mediated Ca²⁺ signaling.

To verify this hypothesis, in vitro yeast 2 hybrid (Y2H) assay was performed to test the interaction between MdSOS2L1 and MdMYB1. Disappointingly, it was found that MdSOS2L1 did not directly interact with MdMYB1 (Fig. 1D). However, in vivo co-immunoprecipitation assay using transgenic apple calli expressing 35S::MdSOS2L1-GFP showed that MdMYB1 protein was immunoprecipitated from 35S::MdSOS2L1-GFP transgenic apple calli cells (Fig. 1E), indicating that MdSOS2L1 and MdMYB1 function as part of a nuclear complex to regulate MdVHA-B1-mediated vacuolar acidification.

To further verify the interaction between MdSOS2L1 and MdMYB1, 35S promoter-driven vectors including MdSOS2L1-RFP and MdMYB1-GFP that express fluorescence-tagged fusion proteins were constructed and used to determine their cellular distribution using an apple calli protoplast system. Upon co-transfection of the MdSOS2L1-RFP fusion gene together with the MdMYB1-GFP fusion gene into the apple protoplasts, the transformant protoplasts were observed in a subcellular localization assay using a laser confocal microscope. The results showed that MdSOS2L1-RFP was co-localized with MdMYB1-GFP in the nucleus, whereas MdSOS2L1-RFP was not co-localized with empty vector GFP in the nucleus (Fig. 1F). This result together with the co-immunoprecipitation assay indicate that MdSOS2L1 interacts with MdMYB1 as part of a nuclear complex to regulate MdVHA-B1-mediated vacuolar acidification.

Currently, we don't know how MdSOS2L1 and MdMYB1 form a nuclear complex to regulate MdVHA-B1-mediated vacuolar acidification. Here, we provide a heroic guess that a nucleus-located protein may directly connect with MdSOS2L1 and MdMYB1 to regulate MdVHA-B1-mediated vacuolar acidification (Fig. 1G).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgements

We thank Prof. Takaya Moriguchi of National Institute of Fruit Tree Science, Japan, for ‘Orin’ apple calli.