*4.4. Gibberellin Content Measurement by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry*

The sample preparation and analysis of GAs were performed according to the method described in [48] with some modifications. Briefly, tissue samples of 26–60 mg DW (three independent technical replicates of each of the tree biological samples) were ground to fine consistency using 3-mm zirconium oxide beads (Retsch GmbH & Co. KG, Haan, Germany) and an MM 301 vibration mill at a frequency of 30 Hz for 3 min (Retsch GmbH & Co. KG, Haan, Germany), with 1 mL of ice-cold 80% acetonitrile, containing 5% formic acid as extraction solution. The samples were then extracted overnight at 4 ◦C using a benchtop laboratory rotator Stuart SB3 (Bibby Scientific Ltd., Staffordshire, UK), after adding 17 internal GA standards ([2H2]GA1, [2H2] GA3, [2H2]GA4, [2H2]GA5, [2H2]GA6, [2H2]GA7) purchased from OlChemIm, Olomouc, Czech Republic. The homogenates were centrifuged at 36,670× *g* and 4 ◦C for 10 min; corresponding supernatants further purified using reversed-phase and mixed mode SPE cartridges (Waters, Milford, MA, USA) and analysed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS; Micromass, Manchester, UK). GAs were detected using multiple-reaction monitoring mode of the transition of the ion [M–H]- to the appropriate product ion. Masslynx 4.1 software (Waters, Milford, MA, USA) was used to analyse the data and the standard isotope dilution method [49] was used to quantify the GAs levels.

#### *4.5. Hormone Treatments*

For hypocotyl and cotyledon measurements, the plants were germinated in vertically oriented square Petri dishes in 22 ◦C, under SD conditions or in continuous darkness as indicated. 6-day-old seedlings were moved to 5 nM IAA (I2886 Sigma, St. Louis, MO, USA)-containing medium for additional 6 days before measured. Epibrassinolid (1 µM; E1641 Sigma) or Ethrel (100 µM; Bayer CropSience, Gent, Belgium) or GA<sup>3</sup> (20 µM; G7645 Sigma) were included into the medium from the beginning of culture. Hormone concentrations not inhibiting hypocotyl elongation were selected based on previous

studies [50–53]. Transgenic RNAi seedlings and corresponding controls were grown in the presence of 5 µM β estradiol in addition to the hormones.

For the gibberellic acid sensitivity assay, constant 60 µE white light was used to limit dark-induced elongation at 22 ◦C. GA<sup>3</sup> was included into the growth medium in concentrations indicated on the figure.

For the complementation of the rosette size, 14-day-old, soil-grown plants were sprayed with 20 µM GA<sup>3</sup> solution supplemented with 0.01% Silwet L-77 (Kwizda, Vienna, Austria). The treatment was repeated at 4-day intervals. The control plants were sprayed with 0.01% Silwet L-77 solution. The GA<sup>3</sup> was dissolved in DMSO:methanol solution (1:1) and stock solutions were prepared at 1 µM (GA3) concentrations for further dilution in water. Rosette size was determined as described earlier, 19 days following the start of the treatment (33-day-old plants).

#### *4.6. Characterization of Mutant*/*Transgenic Plants by RT-PCR*

The Quick-RNA Plant Miniprep Kit (Zymo Research, Irvine, CA, USA) was used to isolate total RNA from whole seedlings. Total RNA was treated by RNase-free DNase I (Thermo Fisher Scientific) and cDNA templates were generated from 0.5 mg RNA samples by RevertAid M-MuLV reverse transcriptase (Thermo Fisher Scientific). The full length transcript of the *RLCK VI\_A2* gene was amplified with primers that were planned for cloning the cDNA in standard PCR reaction (denaturation 94 ◦C for 30 s, annealing temperature 55 ◦C for 30 s, elongation 1 min at 72 ◦C) with DreamTaq polymerase (Thermo Fisher Scientific). *AtGAPC-2* (AT1G13440) transcripts were used as internal reference. See primers in Supplementary Table S7.

Genomic DNA of the GABI\_435H03 T-DNA insertion mutant was isolated with the Phire Plant Direct PCR Kit (Thermo Fisher Scientific) and the T-DNA insertion site was amplified with an At2G18890-specific forward primer (VIA2mid\_F) and a T-DNA-specific reverse primer (T-DNA LB out), according to the supplier's instructions. The purified PCR products were sequenced using the same primers. For the primer sequences, see Supplementary Table S7.

#### *4.7. RNA-Seq and Data Analysis*

The Quick-RNA Plant Miniprep Kit (Zymo Research, Irvine, CA, USA) was used to isolate total RNA from 18-day-old dark-grown seedlings after removing their roots. The RNA preparations were quality checked and quantified using the Agilent RNA 6000 Nano Kit in an Agilent 2100 Bioanalyzer capillary gel electrophoresis instrument (Agilent, Santa Clara, CA, USA). For sequencing library preparation, polyA RNAs were selected from 800 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module, then strand specific indexed libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Libraries were validated and quantified with an Agilent DNA 1000 kit in a 2100 Bioanalyzer instrument, then after pooling and denaturing, library pools were sequenced in an Illumina MiSeq instrument with MiSeq Reagent Kit V3-150 (Illumina Inc., San Diego, CA, USA), generating 2 × 75 bp paired-end reads. Fasq files were trimmed and adapter sequences removed with Trimmomatic 0.33 in paired-end mode [54]. Paired sequences were aligned to the TAIR10 Arabidopsis reference genome using TopHat2 [55]. Binary alignment (\*.bam) files were sorted and deduplicated with SAMtools (http://samtools.sourceforge.net/), then differential expression analysis was done with Cufflinks (http://cufflinks.cbcb.umd.edu/), using Araport 11 transcript annotation [56]. Differential expression was considered as significant with a q value lower than 0.05.

The RNA-seq data used for the analysis have been deposited in the NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) under the accession PRJNA644816.

#### *4.8. Real-Time Quantitative PCR (qRT PCR)*

For qRT PCR, total RNA was purified and converted to cDNA as described under 4.6. The oligonucleotide primers are listed in Supplemental Table S7. A few of them have been previously

published in [53]. qRT-PCR reactions were performed using an ABI PRISM 7700 sequence detection system (Thermo Fisher Scientific) and the qPCRBIO SyGreen Mix Hi-ROX master mix (PCR Biosystems Ltd., London, UK) using standard protocol (denaturation 95 ◦C for 10 min, 40 cycles of 95 ◦C for 10 s, and 62 ◦C for 60 s). Ct values were analysed using the RQ manager software (Thermo Fisher Scientific), and then exported to Microsoft Excel for further analysis. The ratio of each mRNA relative to the mRNA of the *Arabidopsis thaliana UBIQUITIN EXTENSION PROTEIN 1* gene (*UBQ1*, AT3G52590) was calculated using the 2−∆∆CT method. *UBQ1* gene expression was uniform in the wild type and mutant background, as shown by the RNA-seq analysis (see in Supplementary Table S1). The average of the three technical repeats of the WT control was used as reference (unit 1) to calculate relative expression for each gene in the mutant background.

#### *4.9. Statistical Analysis*

Plant culture experiments were carried out in three independent replicates. The number of investigated individuals per replicate is given in each figure legend. Averages with standard errors are shown in the histograms for growth parameters having high and variable sample numbers (e.g., cell, hypocotyl and cotyledon length measurements). In qRT-PCR experiments, two independent biological samples each, with three technical replicates, were amalgamated and analysed together. Student's *t*-test was used for pairwise statistical comparison of the mutant/treated samples to the corresponding wild type/control ones (\* indicates *p*-value < 0.05, \*\* indicates *p*-value < 0.005).

#### **5. Conclusions**

Decreased level or absence of the RLCK VI\_A2 kinase in transgenic Arabidopsis lines resulted in restricted cell expansion and organ/plant size under short day conditions, as well as in continuous dark (skotomorphogenesis), in seedlings as well as in greenhouse plants, indicating the general role of the kinase in plant growth. Transcriptomic analysis confirmed that the kinase might be involved in the modulation of processes that are associated with cell membranes, and take place at the cell periphery or in the apoplast, such as cellular transport and cell wall organisation. Although exogenous GA<sup>3</sup> could rescue the mutant phenotypes, hardly any changes in gibberellin metabolism and/or signalling could be observed in the mutant, indicating that the RLCK VI\_A2 kinase and gibberellin might act parallel on the same/similar processes. To clarify the exact role of the kinase in cell expansion and its hormonal regulation, the identification of its in vivo substrates is required.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/19/7266/ s1. Figure S1: Mapping of transcript sequence reads to the genomic sequence of the At2G18890 gene, Figure S2: Estradiol-induced silencing of the *RLCK VI\_A2* gene, Figure S3: Exogenous hormone treatments, Figure S4: GA<sup>3</sup> complements for the silencing of the *RLCK VI\_A2* gene, Figure S5: qRT-PCR validation of the expression of selected genes, Table S1: Full transcriptomic analysis, Table S2: GO enrichment, Table S3: Hormone-responsive DEGs, Table S4: DEGs overlaping with the DEGs of the *ga1*-3 mutant, Table S5: DEGs directly regulated by the hypocotyl elongation controlling TFs, Table S6: DEGs related to cell wall-related and transport processes, Table S7: Sequences of the used oligonucleotide primers.

**Author Contributions:** Conceptualization, A.F. (Attila Fehér); formal analysis, L.B.; investigation, I.V., E.K., D.T. and A.F. (Anikó Faragó); methodology F.A. and I.D.; resources, A.F. (Attila Fehér); data curation, L.B.; writing—original draft preparation, A.F. (Attila Fehér); writing—review and editing, I.V.; supervision, A.F. (Attila Fehér), M.S. and L.B.; project administration, A.F. (Attila Fehér); funding acquisition, A.F. (Attila Fehér) and M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research including APC was supported by grants from the National Research, Development, and Innovation Office (NKFIH; #K124828) and the Hungarian Ministry for National Economy (GINOP-2.3.2-15-2016- 00001). L.B. was supported by the János Bolyai Research Scholarship (BO/00522/19/8) of the Hungarian Academy of Sciences. The hormone measurements were supported from European Regional Development Fund Project "Centre for Experimental Plant Biology" (No. CZ.02.1.01/0.0/0.0/16\_019/0000738) and the Czech Science Foundation (18-10349S).

**Acknowledgments:** The authors acknowledge the technical support provided by Róza Nagy and Hedvig Majzik (Institute of Plant Biology, Biological Research Centre).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
