The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery

Abstract SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.


Pigments extraction and analysis
Chlorophylls and carotenoids were quantitated by spectroscopic measurements following extraction with 80% acetone from powdered leaves according to Lichtenthaler (1987). For detailed analysis by UPLC, a multi-step extraction procedure was applied as described in Szalonek et al. (2015).
For quantification of carotenoids, α-tocopherol, and plastoquinone chromatograms at 436, 291, and 255 nm, respectively, were integrated using MassLynx 3.5 software (Waters) and results were presented as mol% or mol/mol ratio based on calibration curves obtained for appropriate standards.

Localization of SnRK2.4 and SnRK2.10
Transgenic plants expressing YFP under the native promoter (pSnRK2.4::SnRK2.4-YFP and pSnRK2.10::SnRK2.10-YFP, McLouglin et al., 2012) were used to study the localization of SnRK2.4 and SnRK2.10 kinases in photosynthetic tissue. Plants were grown as described in McLouglin et al., 2012. Leaves from three to four weeks-old plants were detached, infiltrated with water and mounted on slides with homemade chambers. Imaging of YFP fluorophore was carried out on an inverted microscope (Nikon, TE2000) with a confocal laser-scaning mode EZ-C1. YFP fluorescence was excited with blue light emitted by a Sapphire 488 nm laser (Coherent, USA) set at 30% then collected with a 525/40 nm emission filter and displayed in false green. The gain of the photomultiplier was set to 8.9. Since the collected signal was very weak, a wider pinhole size (100 m) was used. Simultaneously the chlorophyll autofluorescence was detected by 610 long pass filter and displayed in false magenta. The gain of the photomultiplier was set to range 6.3-6.9. Palisade mesophyll has been imaged through the epidermis layer using 60x oil immersion objective (Nikon, CFI Plan Apochromat NA 1.4). Single confocal sections and stacks were collected for both lines. The images were digitally processed using FIJI software (NIH, Bethesda, MD, USA) and the figures compiled in Adobe Photoshop 6.0 38 CE (Adobe Systems Inc.).

Stomatal conductance measurements
Six-weeks old plants of wild type and snrk2.10 insertion mutants were grown in hydroponic culture and treated with control media or media supplemented with 150 mM NaCl for up to six days. Stomatal conductance / stomatal aperture in terms of leaf conductance to water vapour was measured with AP4 Leaf Porometer (DeltaT Devices) on fully developed leaves at similar age and expressed in mmol H2O/m 2 /s. For each biological replication three leaves from five plants (n=15) per genotype and treatment were analyzed.

Stomatal index calculation
The 5 th and 6 th leaves were detached from 4-weeks-old plants grown in soil. 0,5cm 2 pieces were cut from the middle leaf blade away from the leaf veins. Pieces from the adaxial and abaxial sides of the leaf were attached to the adhesive tape and the top surface of the tape was immediately covered with a quick-drying nail polish. After 20 min of drying, the top surface was removed and mounted onto a slide. Microscope images of the peeled surface area were taken in several contiguous but not overlapping areas of 0,498 mm -2 (10x objective, Nikon, Eclipse E-800), recorded by a monochromatic camera (ORCA ER, Hamamatsu) and counted in FIJI software (https://imagej.net/contribute/citing, NIH, Bethesda, MD, USA, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855844/). The total number of images collected and counted was 100 for each line. Stomatal and other epidermal cell densities were counted as a number of cells per area unit (mm 2 ). The stomatal index (SI) was calculated as the percentage of the stomata to all epidermal cells, according to the formula: SI = (number of stomata) / (number of total epidermal cells) x100%.

Chlorophyll a fluorescence imaging
Chlorophyll a fluorescence images were recorded using the Maxi version of Imaging-PAM chlorophyll fluorescence system (Heinz Walz, Germany). The A. thaliana plants grown and treated with salt containing or not containing media were dark-adapted for at least 25 min, measurements were performed and plants were returned to the phytotron. The measurements were performed for seven days.
The fluorescence images were recorded with a resolution of 640 × 480 pixels and with the camera parameters set to avoid pixel saturation. Minimal (F0) fluorescence was determined using weak blue modulating light of 0.5 μmol photons m −2 s −1 , whereas maximal (FM) fluorescence was measured through 0.84 s of saturation blue light pulse with 2,700 μmol photons m −2 s −1 . Next, the blue actinic light of 54 μmol photons m −2 s −1 was on, and saturation pulses at 20 s time intervals during 240 s were applied and FM' values were measured. The recorded data were analyzed using ImagingWinGigE software and photosynthetic parameters: FV/FM, Y(II), NPQ, NPQmax, and qL for 5th -7th leaves were calculated. For each leaf, the data were normalized to 1 on day 0 of the control, NaCl, or PEG treatment.

A lack of SnRK2.10 kinase does not affect the changes in abundance and composition of chlorophylls and carotenoid pigments in plants subjected to salinity
Carotenoids are key components of the xanthophyll cycle responsible for protection of unsaturated fatty acids of chloroplast lipids from stress caused by photooxidation (Foyer and Shigeoka, 2011;Horton, 2012;Ashraf and Harris, 2013). A total content of chlorophylls and carotenoids as well as the Chl a/b ratio under stress conditions usually results from the decomposition of chlorophyll-protein complexes. In A. thaliana plants exposed to 150 mM NaCl the content of chlorophylls and carotenoids in dry tissue decreased linearly from day one of exposure, similarly in wt and snrk2.10 plants ( Fig. S8 A, B). Also the Chl a/b ratio decreased linearly during the salt stress, but in the snrk2.10 lines this decrease was apparent sooner than in the wt (  (Fig. S8 K). At the end of cultivation in control conditions the levels of β-potential'. In plants exposed to 150 mM NaCl the content of Neo increased during the time and was higher comparing to control conditions except for the wt plants (Fig. S8 E). In contrast, the Lut level decreased in time during salinity and was lower than in control except for the wt plants (Fig. S8 G). The level of β-Car in all NaCl-treated lines showed similar changes as in control conditions, (Fig. S8 H). However, the Lut/β-Car ratio was unchanged in wt and snrk2.10-3 lines and noticeably decreased in snrk2.10-1 plants at the end of salt treatment comparing to the control conditions (Fig. S8 J). An increase of DEPS was observed in all lines, but was slightly delayed in the mutant lines; at the end of salt treatment, the DEPS values were 3-4 times higher than in control conditions (Fig. S8 I). Similarly, the content of membrane antioxidants PQ and α-T increased rapidly in all examined lines and was two times higher than in the control conditions ( Fig. S8 K).
To sum up, the response of the wt and mutant lines to NaCl treatment was similar and included (i) a decrease of membrane fluidity, (ii) an increase of the membrane 'antioxidant potential', and (iii) an increase of the de-epoxidation state of xanthophylls.

Phosphorylation of LHCII proteins affects phosphorylation of D1 protein under salinity
It has been established that the phosphorylation of PSII and LHCII proteins in Arabidopsis is carried out by two state transition kinases, STN8 (State Transition 8) and STN7 (State Transition 7), respectively. STN8 plays a role in the modulation of the thylakoid ultrastructure phosphorylating mainly PSII core proteins, whereas STN7 is responsible for the maintenance of the redox status of the photosynthetic electron transport chain and counteracts the imbalances in energy distribution between PSII and PSI under unfavorable environmental conditions phosphorylating, among others, the Lhcb proteins (Bonardi et al., 2005;Pesaresi et al., 2011;Longoni et al., 2015). To determine whether the two STN kinases also reciprocally affect the phosphorylation status of each other's substrates under long-term salinity stress we analyzed D1 phosphorylation in an stn7 knockout mutant and the phosphorylation of Lhcb2 in an stn8 mutant. Both mutants displayed an accumulation pattern of D1 and Lhcb2 similar to the wt plants in response to salt stress (Fig. S9 A). As expected, D1 was not phosphorylated in the stn8 mutant and Lhcb2in stn7. Notably, in the stn7 mutant we observed a higher level of D1 phosphorylation than in wt plants indicating that a disturbance in phosphorylation of the LHCII proteins also affects the phosphorylation and probably the turnover of D1 as well in the PSII core. The effect was not reciprocal as the phosphorylation of Lhcb2 was not affected in the stn8 mutant (Fig. S9 A). The STN7 gene expression and STN7 protein level were stable during salinity in wt plants and the snrk2.10 mutants (Fig. S9 B and Fig. S11), suggesting that the increased D1 phosphorylation in snrk2.10 mutants is not related to the amount of STN7 in these plants. Importantly, the expression of STN8 was similar in all plant lines studied (Fig. S11).

Supplemental references
Ashraf M, Harris PJC (2013)  Data are means ± SD from three independent experiments.
Supplemental Figure S8. Effect of salt stress on chlorophyll and carotenoid composition in wt and snrk2.10 plants.
Rosettes from wt and snrk2.10 mutant plants treated (red lines with circles) or not treated (black lines with squares) with 150 mM NaCl for up to six days were ground, pigments were extracted and their content determined as described in Materials and Methods. Content of chlorophyll/carotenoids ratio (D) were calculated basing on pigment absorption spectra and appropriate absorption coefficients. Content of neoxanthin (Neo) (E), a sum of violaxanthin and lutheoxanthin (Vio+Ltx) (F), Lutein (Lut) (G), β-carotene (β-Car) (H), the de-epoxidation status (DEPS) (I), Lut/ β-Car ratio (J) and 'antioxidant potential' (K) were calculated basing on UPLC chromatograms. The abundance of lutheoxanthin, which is converted nonenzymatically from violaxanthin did not exceed 2 mol % and therefore is presented as a sum with violaxanthin. DEPS was calculated as (Z + 0.5A)/(Z + A + V), where Z, A, and V are zeaxanthin, antheraxanthin, and violaxanthin, respectively. 'Antioxidant potential' was calculated as ratio of (α-tocopherol + plastoquinone) to the sum of all detected carotenoids.
The data are mean ± SD for 4 to 9 independent experiments. Statistical significance of difference was determined using ANOVA followed by post hoc Tukeys test (p<0.05). Pairs of results marked with an asterisk differ significantly.     Figure S12. Accumulation of H2O2 in A. thaliana leaves of

snrk2.4 mutant plants under long-term salinity.
Six-week-old plants of wild type and snrk2.4 mutant were treated with 150 mM NaCl or kept in control media for six days. Leaves of the similar age were collected at indicated times and stained for H2O2 with DAB. After removal of photosynthetic and nonphotosynthetic pigments leaves were photographed to show DAB staining. Supplemental Table S1. List of primers used in this study.