SnRK2.6 interacts with phytochrome B and plays a negative role in red light-induced stomatal opening

ABSTRACT

Light is an important environmental factor for plant growth and development. Phytochrome B (phyB), a classical red/far-red light receptor, plays vital role in controlling plant photomorphogenesis and light-induced stomatal opening. Phytohormone abscisic acid (ABA) accumulates rapidly and triggers a series of physiological and molecular events during the responses to multiple abiotic stresses. Recent studies showed that phyB mutant synthesizes more ABA and exhibits improved tolerance to salt and cold stress, suggesting that a crosstalk exists between light and ABA signaling pathway. However, whether ABA signaling components mediate responses to light remains unclear. Here, we showed that SnRK2.6 (Sucrose Nonfermenting 1-Related Protein Kinase 2.6), a key regulator in ABA signaling, interacts with phyB and participates in light-induced stomatal opening. First, we checked the interaction between phyB and SnRK2s, and found that SnRK2.2/2.3/2.6 kinases physically interacted with phyB in yeast and in vitro. We also performed co-IP assay to support that SnRK2.6 interacts with phyB in plant. To investigate the role of SnRK2.6 in red light-induced stomatal opening, we obtained the snrk2.6 mutant and overexpression lines, and found that snrk2.6 mutant exhibited a significantly larger stomatal aperture under red light treatment, while the two independent overexpression lines showed significantly smaller stomatal aperture, indicative of a negative role for SnRK2.6 in red light-induced stomatal opening. The interaction of SnRK2.6 with red light receptor and the negative role of SnRK2.6 in red light-induced stomatal opening provide new evidence for the crosstalk between ABA and red light in guard cell signaling.

Light regulates plant growth and development throughout the life cycle.¹ Besides providing energy for photosynthesis, light also serves as signal to mediate seed germination,² hypocotyl elongation,³ phototropism,⁴ flowering⁵ and stomatal opening.⁶ Plants have evolved several kinds of photoreceptors to perceive the surrounding light signals. As the red/far-red light molecular switch, five phytochromes (phyA-E) in Arabidopsis have been identified, of which phyB has been revealed to play more important role in several physiological processes. For example, phyB mutation leads to smaller stomatal aperture under red light⁷ and hyposensitivity to ABA.⁸

ABA accumulates rapidly when plants are exposed to abiotic stresses including salt, drought, cold, etc., and plays essential roles in the responses to these stresses.^9,10 ABA is perceived by intracellular receptors PYRABACTIN RESISTANCE (PYR)/PYRABACTIN RESISTANCE-LIKE (PYL)/REGULATORY COMPONENT of ABA RECEPTORS (RCAR). In the presence of ABA, those receptors interact with and hence inhibit the activities of clade A PROTEIN PHOSPHATASE 2Cs (PP2Cs), and the inhibition of PP2Cs activities leads to the activation of SnRK2s protein kinases.¹¹ Subsequently, a number of transcription factors, protein kinases, ion channels are activated by SnRK2s to transduce ABA signals.^11,12 Plants lacking ABA signaling molecules, for example, snrk2.2/2.3/2.6 triple mutant, exhibit severe defects in adaption to environmental stresses.¹³

Besides perceiving red/far-red light signals, increasing evidence demonstrate that phyB also participates in ABA response. In tomato, for instance, transcript levels of the key genes responsible for ABA-biosynthesis and signaling were higher in phyB1, phyB2, and phyB1 phyB2 mutants than wild type under red light.¹⁴ In Arabidopsis, phyB mutant is insensitive to exogenous ABA in stomatal conductance, and also leads to a lower expression of ABA-responsive genes.⁸ However, whether ABA signaling components affecting light response in plants remains largely unknown. In this research, we checked the interaction between phyB and SnRK2s and found that SnRK2.2/2.3/2.6 kinases interacted with phyB in yeast cells and in vitro. Since SnRK2.6, also known as OPEN STOMATA 1 (OST1), is involved in regulating stomatal movement,¹⁵ we performed stomatal aperture assay to explore the effects of SnRK2.6 on red light-induced stomatal opening. Results indicate that SnRK2.6 plays a negative role in this process. Coupled with phyB effects on ABA response, these findings suggested a regulatory interplay between light and ABA signaling in guard cells.

SnRK2.2/2.3/2.6 interact with PhyB in yeast cells and in vitro

Red/far-red light receptor phyB participates in the responses to ABA, suggesting that there is a crosstalk between red/far-red light and ABA signaling pathways. Therefore, we investigated the interaction between phyB and SnRK2s, the key regulators in ABA signaling pathways. We first checked the interactions by yeast-two-hybrids (Y2H) assay. There are ten SnRK2s members in the family, namely SnRK2.1 to SnRK2.10. Except for SnRK2.1, the interactions between SnRK2s and phyB were detected. For each pairwise test in yeast, phyB fused to the transcriptional activation domain (AD) of GAL4 served as the prey, and SnRK2s fused to the DNA-binding domain (BD) of GAL4 served as the baits. The results showed that all combinations grew vigorously on SD-Leu/Trp dropout medium (SD-LT), whereas only AD-phyB and BD-SnRK2.2/2.3/2.6 combinations grew on SD-Leu/Trp/Ade/His dropout medium (SD-LTAH), indicating that phyB interacts with SnRK2.2/2.3/2.6 in yeast (Figure 1).

Figure 1.

Analysis of phyB interaction with SnRK2s in yeast

The indicated plasmid combinations were co-transformed into the yeast reporter strain AH109, and interactions of phyB with SnRK2s were assessed by the growth of yeast on medium lacking Leu, Trp, Adenine, and His (SD-LTAH).

To further confirm the interactions between phyB and SnRK2.2/2.3/2.6, we performed in vitro pull-down assay. To obtain green fluorescent protein (GFP)-tagged phyB, protein extracts were prepared from the seedlings of phyB-GFP overexpression line grown in a plant growth chamber for 10 days. SnRK2.2, 2.3 and 2.6 were cloned to pMAL-c2x in-frame with maltose binding protein (MBP) tag. The MBP-SnRKs bound to amylose beads were incubated with phyB-GFP protein extracts. Western blotting results showed that phyB-GFP was pulled down by MBP-SnRK2.2, 2.3 and 2.6, not by MBP alone (Figure 2), suggesting that phyB interacts with MBP-SnRK2.2, 2.3 and 2.6 in vitro.

Figure 2.

The interactions between phyB and SnRK2.2/2.3/2.6 detected by in vitro pull-down assays

Recombinant MBP-SnRK2.2/2.3/2.6 proteins were purified from E. coli using maltose conjugated agarose beads, and were then incubated with supernatant from phyB-GFP overexpression seedlings. The pulled down proteins were detected by western blot using anti-GFP or anti-MBP antibodies. *: nonspecific proteins.

SnRK2.6 is a negative regulator in red light-induced stomatal opening

Interactions between SnRK2s and phyB indicate that SnRK2s might be a potential regulator in red/far-red light response in plants. To test this possibility, we checked the role of SnRK2.6, a guard cell specifically expressed protein,¹⁶ in red light-induced stomatal opening. We first performed stomatal aperture assay with snrk2.6 mutant and found that, after treated with red light for 2 hours, snrk2.6 mutant exhibited larger stomatal apertures than the wild type (Figure 3a), and the difference between snrk2.6 and wild type was significant. Stomatal apertures under red light are smaller than under white light, this may reduce the difference between the mutant and wild type.⁷ Furthermore, SnRK2.2/2.3/2.6 act redundantly in regulating stomatal movements: water loss from leaves of snrk2.2/2.3 was slightly faster than from wild type, but much more quickly from leaves of snrk2.2/2.3/2.6 triple mutant, indicating that SnRK2.2/2.3/2.6 act redundantly in regulating stomatal movements, and SnRK2.6 plays a major role in ABA-induced stomatal closure.¹³

Figure 3.

SnRK2.6 was a negative regulator in red light-induced stomatal opening, and interacts with phyB in plant. (a), snrk2.6 mutant exhibited a significantly larger stomatal aperture than the wild-type plants when treated with red light (Student’s t test, n = 90, *, P<0.05). (b), SnRK2.6 is highly expressed in the overexpression lines. (c), SnRK2.6 overexpression resulted in significantly smaller aperture than the wild-type plants (Student’s t test, n = 90, *, P<.05, **, P<0.01). (d), SnRK2.6 interacts with phyB in vivo. Tobacco leaves co-infiltrated with Agrobacterium GV3101 containing 35S:SnRK2.6-MH and 35S: phyB-GFP or 35S:GFP vectors were subjected to co-IP assay with GFP-trap. The immunoprecipitates were detected with anti-Myc and anti-GFP antibodies, respectively

To gain further evidence to support the role of SnRK2.6 in regulating red light–induced stomatal opening, we obtained several independent transgenic lines, two of them with higher SnRK2.6 expression (20-60 times to Col-0) were used for stomatal aperture assay under red light (Figure 3b). In contrast to the phenotype of snrk2.6 mutant, the two overexpression lines showed significantly smaller stomatal apertures than the wild type after 2 hours red light treatment (Figure 3c). Combined with the phenotype of snrk2.6 mutant in response to red light, these results supported that SnRK2.6 plays a negative role in regulating red light-induced stomatal opening.

Since SnRK2.6 is involved in regulating red light-induced stomatal opening, we then checked whether SnRK2.6 interacts with phyB in vivo. Tobacco leaves were infiltrated with Agrobacterium GV3101 strain harboring phyB-GFP and SnRK2.6-Myc, and cell lysates were immunoprecipitated with GFP-trap and detected with anti-GFP and anti-Myc antibodies. The coimmunoprecipitation (co-IP) results showed that immunoprecipitation of phyB-GFP pulled down SnRK2.6-Myc (Figure 3d). These results provided evidences that SnRK2.6 interacts with phyB in plant, and plays important roles in red light induced stomatal opening.

Discussion

In well-watered conditions, light promotes stomatal opening to facilitate photosynthesis at the expense of a higher water loss.¹⁷ When the available water become scarce, plants close the stomata to reduce water loss. ABA plays a pivotal role in stomatal closure in response to environmental stress. Recent studies revealed that there is a crosstalk between signaling pathways of light and ABA. For instance, expression of PYL5, an ABA receptor expressed in stomata, and RAB18 and RESPONSIVE TO DESICCATION 29A (RD29A), two ABA-responsive genes, are significantly decreased in phyB mutant when treated with ABA.⁸ phyB mutant synthesizes much more ABA than wild type in well-watered conditions in Arabidopsis and tobacco, and exhibits enhanced salt and drought tolerance.^8,18 Most recently, Holalu et al. report that higher ABA levels in phyB mutant is due to PHYTOCHROME INTERACTING FACTOR 4/5 (PIF4/PIF5) accumulation.¹⁹ The expression of the ABA biosynthetic gene NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) is elevated in phyB mutant, but suppressed in the phyB pif4 pif5 triple mutant, indicating that PIF4 and PIF5 enhance NCED3 transcription and ABA accumulation. Reduced ABA accumulation in the phyB pif4 pif5 triple mutant results in an increased branching, which is controlled by BRANCHED 1 (BRC1) gene.¹⁹ In phyB mutant, the higher level of PIF4 and PIF5 lead to the higher expression of BRC1, which in turn accelerates NCED3 expression and ABA accumulation.²⁰ In addition, Qi et al. found that PIF1/3/4/5 directly bind to the G-box motif in ABA INSENSITIVE 5 (ABI5) promoter and enhance ABI5 transcription, which resulting in reduced ABA sensitivity during seed germination.²¹ PIFs also interact with ABA receptors PYL8 and PYL9 to modulate ABA response.²¹

The decreased expression of the key genes in ABA signaling and increased ABA accumulation in phyB mutant indicate that phyB, together with some molecules in red-light signaling, affects ABA response with different mechanisms. Considering the important roles of phyB and ABA in plant development and abiotic stress responses, there should be more molecules involved in the crosstalk between light and ABA signaling. Here in this study, we found that SnRK2.2/2.3/2.6, the core components of ABA signaling, interacted with red-light receptor phyB in yeast and in vitro, and SnRK2.6 interacts with phyB in plant. ABA inhibits light-induced stomatal opening, and the calcium, hydrogen peroxide and nitric oxide mediate ABA inhibition of stomatal opening.^22,23 It has been shown that SnRK2.6 prevents stomatal opening in the presence of ABA by inhibiting the activity of KAT1, an inward K⁺ channel in guard cells.²⁴ Here, we show that SnRK2.6 interacts with phyB and plays a negative role in red light-induced stomatal opening. It is possible that phyB mediates red light-induced stomatal opening by at least three ways: 1) increases the osmolytes in guard cells by enhancing photosynthesis;²⁵ 2) interacts with PIF4/5 to inhibits ABA biosynthesis by repressing NCED3 expression; 3) triggers a negative mechanism to prevent excessive stomatal opening: interacts with and increase SnRK2.6 kinase activity, which lead to the repression of K⁺ influx and inhibition of stomatal opening.

In brief, results in this research suggest that both SnRK2.6 and phyB are involved in regulating red light induced stomatal movements. SnRK2.6 interacts with phyB and plays a negative role in this process. The negative mechanism triggered by SnRK2.6 will help to fine-tune the stomatal aperture under red light, which will improve the water use efficiency.

Material and methods

Plant materials and stomatal aperture assay

The plant materials used in this study were Arabidopsis (Arabidopsis thaliana) with the Col-0 background. snrk2.6 (SALK_008068C), a T-DNA insertion line with faster water loss,¹³ was obtained from the Arabidopsis Biological Resource Center (ABRC). SnRK2.6 overexpressing lines were obtained according to the methods described previously.²⁶ In brief, the coding region of SnRK2.6 was amplified from wild-type cDNA, and then integrated into the pCAMBIA1300 vector under the control of CaMV 35S promoter. The 35S:SnRK2.6-pCAMBIA1300 vector was transformed into wild-type Arabidopsis by the floral dipping method.²⁷ The homozygous hygromycin-resistant lines were selected in the T3 generation.

For stomatal aperture assay, seeds were first sterilized and then planted on half-strength MS medium. Plates were kept in 4°C for two days to facilitate germination, and then were moved to a plant culture chamber at 22°C with a 16-h-light/8-h–dark cycle. Ten days later, seedlings were transplanted into soil, and grown under the same conditions for 2–3 weeks. Stomatal aperture assays were performed essentially as previously described.²⁶ In brief, the leaves were incubated in the dark for 1 h to close the stomata in MES buffer (30 mM KCl, 0.1 mM CaCl₂, 10 mM MES, pH 6.1), and then the epidermis was peeled and illuminated with red light (50 μmol m⁻² s⁻¹) for 2 hours. Red light (660 nm) was supplied by LED lamps (22–24 V, 1500 mA; HOUKEM, China). The stomatal apertures were measured under a microscope. Thirty stomata were selected randomly on 5-6 leaves for three independent replicates before or after light treatments. The data are presented as the means ± SD (n = 90), and significance was analyzed by Student’s t test.

Yeast two hybrid (Y2H) assay

Y2H assay was performed according to the previously described.²⁶ In brief, all the bait and prey combinations were co-transformed into AH109 competent cells, and incubated on synthetic dropout medium lacking Leu and Trp (SD-LT) for 4 to 5 d at 30°C. The transformants were then spotted onto synthetic dropout medium lacking Leu, Trp, Ade, and His (SD-LTAH), and colony growth was photographed after 4 to 5 d. All the experiments were repeated at least three times.

In vitro pull-down assay

phyB proteins were obtained from phyB-GFP overexpression Arabidopsis plants. About 0.5 g seedlings grown in the chamber with a 16-h-light/8-h-dark cycle for about 10 days were harvested and ground to fine powder in liquid nitrogen, and suspended with 1 mL immunoprecipitation buffer,²⁶ vortexed thoroughly and then rotated slowly at 4°C for 1 h. Then centrifuged at 14000 g for 15 min at 4°C, the supernatant was ready for pull-down assay.

To obtain recombinant proteins, the coding sequences of SnRK2.2, SnRK2.3 and SnRK2.6 were amplified from total cDNA using the primers shown in Supplemental Table S1, and cloned into pMAL-c2x vector, and the constructs were then transformed into BL21 (DE3) cells. SnRK2.2/2.3/2.6 fusion proteins were purified with maltose agarose beads (New England BioLabs Inc.). After washing thoroughly, equal amounts of MBP or MBP-SnRK2.2/2.3/2.6 on agarose beads were incubated with plant supernatant from phyB-GFP overexpression lines for 2 hours at 4°C. All precipitates were washed three times with immunoprecipitation buffer, and boiled with 2× SDS sample buffer for 5 min. The pulled down proteins were detected by western blot using anti-GFP or anti-MBP antibodies.

In vivo co-IP assay

To obtain the 35S:phyB-GFP and 35S:SnRK2.6-Myc vectors, coding sequences of phyB and SnRK2.6 were integrated into modified pCAMBIA1300 vectors containing CaMV 35S promoter and GFP or Myc flags. Primers used for vector construction were listed in Supplemental Table S1. Tobacco leaves infiltrated with Agrobacterium GV3101 containing those two vectors were used for checking phyB and SnRK2.6 interaction, and the leaves infiltrated with 35S:GFP and 35S:SnRK2.6-MH containing Agrobacterium were used as the control. Total protein was extracted with immunoprecipitation buffer as mentioned above. Equal amounts of total protein were then incubated with GFP-trap for 3 hours at 4°C. The precipitate proteins were detected by western blot using anti-GFP or anti-Myc antibodies.

Funding Statement

This work was supported by the National Science Foundation of China [32070191 to Y.-L.C.], Natural Science Foundation of Hebei Province [C2019205168 to C.-G.Z.], and Scientific Research Foundation of Hebei Province for the Returned Overseas Chinese Scholars (to C.-G.Z.)

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website