Phosphorylation of plasma membrane aquaporin PIP2;1 in C-terminal affects light-induced stomatal opening in Arabidopsis

ABSTRACT

Guard cells undergo quick volume changes during stomatal movements. However, the contribution of aquaporins to stomatal movements has not been well understood. The plasma membrane aquaporin PIP2;1in Arabidopsis has been found to mediate abscisic acid–induced or flag22-induced stomatal closure. In this research, we investigated the role of PIP2;1 in light-induced stomatal opening by measuring the stomatal apertures of the pip2;1 mutant and PIP2;1 overexpression lines after light treatment. pip2;1 mutant exhibited a larger stomatal aperture, and the overexpression lines displayed a smaller stomatal aperture. It has been reported that the phosphorylation at Ser-280 and Ser-283 of PIP2;1 in rosette tissue increased in response to darkness, whereas osmotic water permeability (P_f) in mesophyll protoplasts in darkness was lower than that under light, suggesting that phosphorylation at Ser-280 and Ser-283 of PIP2;1 affected P_f in mesophyll protoplasts. Therefore, we obtained the pip2;1 mutant expressing phosphorylation-deficient (PIP2;1 AA) or phosphomimetic (PIP2;1 DD) forms of PIP2;1. The PIP2;1 AA lines exhibited a larger stomatal aperture as pip2;1 mutant, whereas PIP2;1 DD lines exhibited a smaller stomatal aperture as PIP2;1 overexpression lines under light. These results suggest that PIP2;1 plays a negative role in light-induced stomatal opening, and phosphorylation of PIP2;1 at Ser-280 and Ser-283 causes reduced water absorption in guard cells and decreased stomatal opening.

KEYWORDS:

• Light
• stomatal opening
• plasma membrane aquaporin PIP2
• 1
• phosphorylation

PIP2;1 plays a negative role in light-induced stomatal opening

Stomata surrounded by a pair of guard cells control the gas exchanged between plants and environment. Stomatal movements are modulated by several endogenous and environmental signals, including phytohormone abscisic acid (ABA), jasmonic acid (JA), extracellular ATP, CO₂, humidity, and light/darkness.^2–6 Both blue light and red light induce stomatal opening by increasing K⁺ and sugar contents in guard cells, which provide the osmotic potential for water absorption.^7,8 Guard cells can change their volume by up to 40% in stimulus-induced stomatal movements in a few minutes.⁹ However, the mechanisms of water transport across the membrane during stomatal movements have not been clearly understood. Plasma membrane intrinsic proteins (PIPs) in plants represent the most abundant aquaporins in the membrane,¹⁰ which facilitate water diffusion across the membrane. Several PIPs have been found to be expressed in guard cells.^11,12The role of PIPs in stomatal movements was uncovered in several species. Constitutively, overexpression of a grape VvPIP2;4 N led to a higher stomatal conductance and gas exchange under well-watered condition, whereas it caused a decrease in stomatal conductance and leaf gas exchange with water stress.¹³ There is divergence on the roles of PIP2;1 in stimulus-induced stomatal closure in the reports from different groups; although it was reported that the mutation in AtPIP2;1 alone did not affect CO₂-induced or ABA-induced stomatal closure,¹⁴ two reports from other groups showed that AtPIP2;1, phosphorylated at Ser-121 by OST1 or BAK1, mediated ABA- or flag22-induced stomatal closure.^15,16To explore the role of PIP2;1 in light-induced stomatal opening, we analyzed the stomatal aperture of the widely used pip2;1-null mutant^15,17 upon white light illumination. Unexpectedly, the pip2;1 mutant exhibited a larger stomatal aperture after 1 or 2 h of light illumination, suggesting that PIP2;1 plays a negative role in light-induced stomatal opening (Figure 1b). To gain further evidence to support the role of PIP2;1 in light-induced stomatal opening, we obtained two independent PIP2;1 overexpression lines with high expression levels of PIP2;1 (Figure 1a). In contrast to the phenotype of the pip2;1 mutant, the two PIP2;1 overexpressing lines showed significantly smaller stomatal apertures after 1 or 2 h of light treatment (Figure 1b), further supporting the negative role of PIP2;1 in light-induced stomatal opening. The difference between pip2;1 and wild type in stomatal aperture after 2 h of light treatment became more significant than that with 1 h of light treatment; therefore, we measured the stomatal apertures after 2 h of light treatment in the next part. It has been reported that pip2;1 mutant exhibited similar stomatal opening as wild type¹⁵; the larger stomatal aperture of pip2;1 after light illumination found in this research is a variance of the phenotype, which may be due to the variation of the plant growth condition or the subtle difference in stomatal assay procedure.

Figure 1. PIP2;1 play a negative role in white light-induced stomatal opening. (a) Expression of PIP2;1 in pip2;1 mutant and two PIP2;1 overexpression lines. (b) Stomatal apertures of pip2;1 mutant and the PIP2;1 overexpression lines (OE-37and OE-50) after 1 or 2 h of white light treatment (*, P < 0.05; **, p < 0.01).

Phosphorylation at Ser-280 and Ser-283 in C-terminal of PIP2;1 affects light-induced stomatal opening

The smaller stomatal apertures of the PIP2;1 overexpression lines caused by reduced water absorption may be due to post-translational modification under light. It has been reported that osmotic water permeability (P_f) of mesophyll protoplasts in darkness was lower than that under light (Figure 3a in the study by Prado et al. [1]), and the phosphorylation Ser-280 and Ser-283 in the C-terminal of PIP2;1 in rosettes was higher in darkness than that under light (Figure 5a in the study by Prado et al. [1]), suggesting that there is a possible link between the decrease of mesophyll protoplasts P_f and the phosphorylation at Ser-280 and Ser-283 in PIP2;1. To investigate whether the reduced water uptake in PIP2;1 overexpression lines is due to the phosphorylation at Ser-280 and Ser-283 in PIP2;1, we mutated Ser-280 and Ser-283 to Ala (the phosphorylation-deficient form, PIP2;1 AA) or Asp (the phosphomimetic form, PIP2;1 DD), respectively. To avoid the transcriptional regulation of PIP2;1 expression and the role of the endogenous wild-type PIP2;1 protein, we introduced PIP2;1 AA or PIP2;1 DD driven by a 35S cauliflower mosaic virus promoter into pip2;1 mutant. We identified the transgenic plants with the high expression level of PIP2;1 AA or PIP2;1 DD (Figure 2a) and measured the stomatal apertures of these lines illuminated with white light. The results showed that the two independent PIP2;1 AA lines exhibited larger stomatal apertures as pip2;1 mutant, whereas the two PIP2;1 DD lines showed smaller stomatal apertures as PIP2;1 overexpression lines (Figure 2b). The stomatal response of PIP2;1 overexpression lines is consistent with that of the PIP2;1 DD overexpression lines, suggesting that the reduced water uptake is likely due to the phosphorylation of Ser-280 and Ser-283 in C-terminal of PIP2;1. The higher difference in the relative transcription level of PIP2;1 under the control of 35S promoter in transgenic lines did not lead to a stomatal aperture variation (Figure 2), which may attribute to the similar protein levels in these lines. When guard cells have sufficient PIP2;1 proteins, the translation from mRNA to protein may be inhibited. Therefore, the transgenic lines with different expression levels of mRNA may have similar protein levels and exhibit similar phenotype.

Figure 2. Phosphorylation at Ser-280 and Ser-283 of PIP2;1 is responsible for the negative role of PIP2;1 in white light-induced stomatal opening. (a) The expression level of PIP2;1 (AA or DD forms) in the corresponding lines. (b) Stomatal apertures of PIP2;1 AA and PIP2;1 DD lines with 2 h of white light illumination (**, p < 0.01).

The smaller stomatal apertures in PIP2;1 overexpression lines after light treatments suggest that the water uptake in guard cells of these lines is inhibited under light. The PIP2;1 DD overexpression lines resembled the wild-type PIP2;1 overexpression lines in stomatal opening after light illumination, suggesting that the inhibition of water absorption is possibly due to the phosphorylation at Ser-280 and Ser-283 in PIP2;1. In the pip2;1 mutant, release of the inhibition caused more water absorption and a larger stomatal aperture after light treatment. Therefore, due to the different phosphorylation sites, PIP2;1 plays a different role in ABA- or light-induced stomatal movements; when PIP2;1 is phosphorylated at Ser-121, it plays a positive role in ABA-induced stomatal closure; when PIP2;1 is phosphorylated at Ser-280 and Ser-283, it plays a negative role in light-induced stomatal opening. H₂O₂ is an important signaling molecule involved in the regulation of stomatal movements. It has been shown that H₂O₂ induces stomatal opening at low concentrations and inhibits stomatal opening at high concentrations. Light triggers an increase of H₂O₂ level in guard cells, and H₂O₂ at the cellular level enhances stomatal opening.¹⁸ Light also elevates H₂O₂ level in mesophyll cells by photorespiration and electrotransfer during photosynthesis.^19,20 PIP2;1 facilitates the entry of H₂O₂ into guard cells in ABA- or pathogen-triggered stomatal closure.16 Therefore, it is possible that the transport of H₂O₂ from mesophyll to guard cells in PIP2;1 or PIP2;1 DD overexpression lines under light is higher than that of wild type, which causes partial inhibition of stomatal opening. This may be another reason for the reduced stomatal opening response in PIP2;1 or PIP2;1 DD overexpression lines after light illumination.

Materials and methods

Plant materials and growth conditions

The Arabidopsis (Arabidopsis thaliana) used in this study were the Col-0 background. Seedlings were grown in a greenhouse under long-day conditions [16-hlight/8-hdark cycle), with a photon flux density of 150 μmol m⁻² s⁻¹ and a temperature from 18°C to 22°C. The mutated PIP2;1 AA and PIP2;1 DD cDNA were generated by Polymerase Chain Reaction (PCR) using mutagenic primers according to Prado et al. 1. Then, PIP2;1, PIP2;1 AA and PIP2;1 DD cDNA were integrated into the pCAMBIA1300 vector, respectively. These constructs were transformed into Arabidopsis using the floral dipping method.²¹ The transformants were selected in MS media containing hygromycin (50 mgL⁻¹). The expression levels of PIP2;1 in these transgenic lines and pip2;1-null mutant [SM_3_35928, 15, 17] were checked by qRT-PCR with the primers F:5ʹ- TCCAATCGGATTTGCCGTGT-3ʹ and R:5ʹ-GTCATCCCATGGCTTGCTCT −3ʹ.

Stomatal aperture assays under light treatments

Stomatal aperture measurements were performed essentially as described by Wang et al. 22. In brief, fully expanded rosette leaves of 3- to 4-week-old plants were used for the stomatal assay. To close the stomata, the leaves were collected and incubated in 2-(N-morpholino) ethanesulphonic acid (MES) buffer (10 mM MES, 30 mM KCl, 0.1 mM CaCl₂, pH 6.1) in the dark for 1 h, and then, the epidermis was peeled and illuminated in MES buffer with white light (150 μmol m⁻² s⁻¹) for the indicated time in figure 1 and 2. The stomatal apertures were determined under a microscope. Fifty stomata were randomly selected for three independent replicates before or after light treatments. The data are presented as means ± SE (n = 150).

Disclosure statement

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by the National Science Foundation of China [31571450 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.)