Arabidopsis CK2 family gene CKB3 involved in abscisic acid signaling

CKB3 is a regulatory (beta) subunit of CK2. In this study Arabidopsis thaliana homozygous T-DNA mutant ckb3 was studied to understand the role of CKB3 in abscisic acid (ABA) signaling. The results shown: CKB3 was expressed in all organs and the highest expression in the seeds, followed by the root. During seed germination and root growth the ckb3 mutant showed reduced sensitivity to ABA. The ckb3 mutant had more stomatal opening and increased proline accumulation and leaf water loss. The expression levels of number of genes in the ABA regulatory network had changed. This study demonstrates that CKB3 is an ABA signaling-related gene and may play a positive role in ABA signaling.


Introduction
Plants are protected by the expression of various stress-related genes to synthesize hormones and regulatory plant growth and development as well as mediators of environmental stress responses (Sreenivasulu et al., 2012;Gnutt et al., 2017). Among various phytohormones abscisic acid (ABA) is the major hormone which is the central regulator of abiotic stress resistance in plants and coordinates an array of functions (Finkelstein, 2013;Wani and Kumar, 2015), enabling plants to cope with different stresses. A previous study shown that ABA acts as a stress signal in plants and plays an important role in modulating plant response to various biotic and abiotic stresses including cold drought salinity stress and so on (Ma et al., 2019;Wang et al., 2019). Genetic and chemical studies exemplifying ABA in regulating seed maturation and dormancy are important as they have showed that a reduction this hormone level is associated to decreasing seed dormancy (Li et al., 2012;Lee et al., 2015).
CKB3 is a regulatory (beta) subunit of CK2 involved in regulation of the circadian clock in Arabidopsis (Sugano et al., 1999). Plant hormone and stress-response elements were found through gene chip analysis, including ABRE (ABA-responsive element), AuxRE (auxin-responsive element), CGTCA-motif (MeJA-responsive element) and HSE (Heat-responsive element). Thus the expression of CKB3 gene was also regulated by hormones and stresses and that it might play an important role in hormone and stress-response pathways.
In this study to understand the role of CKB3 in abiotic stress signaling Arabidopsis thaliana homozygous T-DNA mutant ckb3 was used. The physiological and biochemical indicators were measured, such as the germination, root growth, hypocotyl elongation, stomatal apertures, water-loss rate and so on, then combined the expression of CKB3 gene in response to various stresses of T-DNA mutant ckb3 and Col-0to analysis the role of CKB3 in abiotic stress. The results provide a basis for further study of CKB3 involved in various stress responses.

Identification of homozygous T-DNA insertion mutants
The Arabidopsis thaliana Columbia wild-type (Col-0) was used as an Arabidopsis wild-type. From the Arabidopsis Biological Resources Center (ABRC) purchased the T-DNA insertion mutants ckb3 (Salk_093548 with Col-0 as background). Using tri-primer-PCR method to identify homozygous T-DNA insertion mutants, and the primers information were listed in Table 1. The primers were provided by ATIDB (the Arabidopsis thaliana Integrated Database).

Germination assays and root growth
To surface-sterilize the seeds for germination assay, using 75% ethanol to wash the seeds for 30 s followed by 20% NaClO for 10 min, then washed the seeds six to ten times with sterile distilled water and then placed in 4 °C for vernalization. The seeds were planted on Murashige and Skoog (MS) medium that contained 3% sucrose and 8% agar (PH = 5.8-6.0) with different concentrations of ABA (0 μM, 0.3 μM, 0.6 μM, 1 μM) after 4 days and then transferred to a growth chamber at 22 °C with about 80 μmol photons m -2 s -1 under 16 h of light/ 8 h of dark and 60% relative humidity. About root elongation analysis, the seeds were surface-sterilized using the above methods. The seedings were grown in MS plates for 3 days and then transferred to ABA-containing (10 μM, 40 μM) or ABA-free MS medium and continued to grow for 6 days before measurement.

Stomatal aperture measurement
Using rosette leaves of 4-week-old plants to measure the stomatal aperture. To incubate the detached leaves in solution containing 10 mM MES, 50 mM KCl and 10 mM CaCl 2 (pH 6.15) for 2 h under light. Then add ABA to the solution to the final concentration was 1 M. After the detached leaves were treated for 2 h, the stomatal apertures were measured as described previously (Sun et al., 2012).

Determination of the water-loss rate
To detect plant water-loss rate used the method was described by Shan et al. (2012). Getting the rosette leaves from an approximately 3-week-old T-DNA insertion mutants and Col-0 plants to detect the water-loss rate. Put the detached rosette leaves in clean filter paper, and then placed into a growth chamber with 25 °C and humidity of 60%. To recorded the fresh weight every 30 min. Each experiment was repeated three times.

Proline content measurement
To incubate the 14-day-old seedlings of Col-0 and T-DNA insertion mutants in MS solution containing 3% sucrose and 8% agar (PH = 5.8-6.0) with different concentrations of ABA (0 or 100 μmol). The 14-day-old seedling plants were collected and using the sulfosalicylic acid method to extract proline (Qin et al., 2014). The experiment was performed in triplicates.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the 14-day-old Col-0 and mutant with TriZol (Takara) which was incubated in an ABA solution. To synthesize the first strand cDNA using the Maxima  First cDNA Synthesis Kit (Fermentas). Using a SYBR  Green I kit (TOYOBO, Japan) to perform quantitative PCR (qRT-PCR) in an Mx3000P thermal cycler (Stratagene USA). The procedure of the PCR reactions was started with a denaturing step for 10 min at 95 °C followed by 50 cycles of 15 s at 95 °C and a primer extension reaction at 55 °C for 1 min. The ACTIN2 gene was used as an internal control. All qRT-PCR tests were run in duplicates each with three biological replicates. The primers information was listed in Table 2. Analyze the data using MxPro (Stratagene) software.

CKB3 T-DNA insertion mutants homozygous identification
Through PCR and qRT-PCR technology to identify the CKB3 T-DNA insertion mutants homozygous plants.
The flanking region sequences of the T-DNA insertion mutant ckb3 showed that the insertion was located 80 bp

Primer name
Primer sequence 5'-3' TTCCAACTCCTCTTGCTCCT downstream of the ATG start codon and inverted insertion.
In the end we got three individuals homozygous of T-DNA insertion mutant ckb3 ( Figure 1B). Through qRT-PCR technology to analyze the expression of homozygous of T-DNA insertion mutant ckb3. The result showed that the expression of CKB3 gene in T-DNA insertion mutant plant was zero indicating that the T-DNA insertion severely impaired the CKB3 gene expression.

Expression of the CKB3 gene in different organs and in response to various stresses
The expressions of CKB3 gene were analyzed in roots, stems, rosette, cautine leaves, flowers, silique and seeds of Arabidopsis ( Figure 1D). CKB3 was expressed in all organs and the highest expression in the seeds, followed by the root. To analyze the expression of CKB3 gene under different stresses, the seedlings of 14 days old were Table 2. Primer sequences used in quantitative RT-PCR.

Primer
Primer sequence 5'-3' treated with ABA, GA, IAA and NaCl for different lengths of time (Figure 2). The expression of CKB3 peaked when treated by ABA for 6 h, IAA stress for 1 h. The expression decreased at first and then increased with time when treated by GA and NaCl. The results indicated that CKB3 may participate in the ABA stress signaling pathway.

CKB3 is involved in the ABA-mediated inhibition of seed germination and root elongation
CKB3 gene has the higher expression in seeds and roots than the other organ, to certain the effect CKB3 on seed germination and root growth. The seeds of T-DNA insertion mutant ckb3 and Col-0 were sown in a MS medium added with different concentrations of ABA after surface disinfection. The seed germination data was checked from 1 day to 7 day. The T-DNA mutant ckb3 displayed a higher germination rate than the wild-type Col-0 (Figure 3) in the presence of 0.3 μmol/L, 0.6 μmol/L and 1μmol/L ABA. The ckb3 was less sensitive to ABA than the wild type. When treated with 10 μM ABA or 40 mM NaCl, the ckb3 had apparently higher root growth than Col-0 ( Figure 4A). These results were match to the seed germination assay.

CKB3 gene affects stoma aperture, water loss and proline contents under ABA-mediated
Based on previous research results, in order to survive harsh conditions the plants can produce ABA to change stomatal openness, water loss and proline content (Schroeder et al., 2001;Verslues and Bray, 2006;Seiler et al., 2014;Eisenach et al., 2017). The results showed that the stomatal apertures of ckb3 were larger than those of the wild type with ABA treatment (10 μmol/L ABA) ( Figure 4B). These results indicated that CKB3 might play a negative role under the influence of ABA in ABA-regulated stomatal closure. In this study shown the leaves of T-DNA insertion mutant ckb3 lost water at a slower rate than the Col-0 leaves ( Figure 4C). To certain whether CKB3 affects proline accumulation in plants in response to ABA, the proline contents of the wild type ckb3 plants in response to ABA was determined. As shown in Figure 4D, the T-DNA insertion mutant ckb3 had significantly higher accumulated proline than Col-0. Thus, under the influence of ABA, CKB3 aslo plays a negative role.

CKB3 regulates the expression of ABA and stress responsive genes
In order to confirm whether CKB3 is involved in the ABA signaling pathway, expression levels of ABA signaling pathway related genes ABI3, ABI5, ABF2, OST1, RAB18 and EM1 (Yoshida et al., 2015;Skubacz et al., 2016;Gao et al., 2016;Wang et al., 2018). As shown in Figure 5, the expression level of ABI3 in T-DNA mutant plant was much lower than this in Col-0, but the expression levels were equal in ckb3 T-DNA mutant and Col-0 with ABA treatment displayed. The expression levels of ABI5, OST1, ABF2 and EM1 in the ckb3 plants were lower. These results showed that ( Figure 5) CKB3 gene can regulate the expression level of ABA and stress-related genes, indicating that CKB3 may positively affect the ABA signaling.

Discussion
A previous have shown that several CK family genes are involved in the ABA signaling pathway, such as that three nuclear-located CK2 α-subunits (CKA1-3) in Arabidopsis have a synergistic role in ABA-induced blockage (Mulekar et al., 2012). CKA4 gene is an enhancing factor in abiotic stress signalling through modulating the expression of some molecular players in retrograde signaling (Wang et al., 2014). CKB1 is involved in abscisic acid to regulate stress responses in Arabidopsis thaliana (Yuan et al., 2017). Previous studies have shown that CKB3 gene is a key component of the plant circadian clock system, including the long-day plant Arabidopsis, there is no reports on the involvement of this gene in stress response. Through gene chip analysis we found certain plant hormone and stress-response elements, such as ABRE (ABA-responsive element), AuxRE (auxin-responsive element), CGTCA-motif (MeJA-responsive element) and HSE (Heat-responsive element). In this study to understand the role of CKB3 in abiotic stress signaling Arabidopsis thaliana homozygous T-DNA mutant ckb3 was used. The germination, root growth, hypocotyl elongation, stomatal apertures, water-loss rate, proline content and the expression of CKB3 in response to ABA of T-DNA mutant ckb3 and Col-0 were measured. These results can determine whether CKB3 gene is involved in the ABA signaling pathway.Although expression of CKB3 in all organs, its expression increased in roots and seeds and this result agrees with the expression of CKB1 gene (Yuan et al., 2017). The ckb3 mutant showed reduced sensitivity to ABA during seed germination and seedling growth more stomatal opening and increased proline accumulation, these results indicate CKB3 may be play a negative role in regulating seed germination, seedling growth, stomatal opening and proline accumulation under the influence of ABA. ABI3 (ABA Insensitive 3) plays a negative feedback regulatory role in seed germination (Reyes and Chua, 2007). ABI5 (ABA Insensitive 5) is a basic leucine zipper transcription factor that plays a pivotal role in the regulation of early seedling growth and seed germination in the abiotic stresses and ABA (Skubacz et al., 2016). Without ABA treatment the expression analysis of stress-responsive genes showed that the expressions of ABI3 and ABI5 were lower in CKB3 T-DNA mutants plants than in Col-0 plants, the expression ABI3 was equal in T-DNA mutants ckb3 and Col-0 and ABI5 was lower in CKB3 T-DNA mutants plants than in Col-0 plants when treatment with ABA.
These results shown CKB3 plays a role in regulating seed germination under the influence of ABA. OST1 is well characterized at molecular and physiological levels  to control stomata closure in response to water-deficit stress (Xuanyuan et al., 2017). After ABA processing the OST1 expression was lower in the T-DNA mutant ckb3 than in the Col-0 plants. The expression of ABF2 (ABRE binding factor 2) and EM1 was similar to the level of OST1. The expression of ABF2 has been reported to be strongly induced by salt, drought and ABA (Zandkarimi et al., 2015;Zhou et al., 2016). EM1 (Early Methionine-Labeled) was ABA-related gene the expression can be induced by ABI5 (Skubacz et al., 2016). These results indicate that CKB3 is an ABA signaling related gene and future is to explore whether this gene is involved in the plant stress response.