**1. Introduction**

Eukaryotic protein kinases form a large superfamily, and are associated essentially with all cellular functions. During evolution, protein kinase families have evolved independently in the lineages of eukaryotes. In consequence, the various kinase families are unevenly represented in the different eukaryotic organisms, which also have specific kinase classes. The number of protein kinase coding genes is especially high in plant genomes. In *Arabidopsis thaliana*, ~4% of the protein coding genes code for protein kinases while this percentage is ca. 2% in *Homo sapiens* [1]. This high number of protein kinases is likely due to the importance of cell-to-cell communication during the post-embryonic development of plants that is strongly influenced by the environment. In addition, plant defence and immunity depend on the specific recognition of pathogen-associated molecular patterns. In plants, cell-to-cell communication as well as innate immunity rely on plant receptor-like kinases (RLKs), which account for more than half of protein kinases in Arabidopsis (>600 RLK genes out of >1000 kinase-coding genes). RLKs resemble the receptor kinases of animals. The sequence of their kinase domain indicates that they are rather related to the animal cytoplasmic Pelle and interleukin receptor-associated kinases [2]. Moreover, RLKs exhibit serine/threonine kinase specificity in contrast to animal receptor kinases that are almost exclusively tyrosine kinases. This indicates that ancient RLK/Pelle kinases were co-opted for transmembrane signalling in plants after their divergence from animals.

RLKs can be classified into several families based on their various extracellular ligand-binding and slightly divergent cytoplasmic kinase domains [2]. There are also a number of RLK-like kinases that have only cytoplasmic kinase domain but no extracellular ligand-binding domain (only a few of them have a transmembrane domain) [3]. These cytoplasmic protein kinases are referred to as receptor-like cytoplasmic kinases, or receptor-like cytoplasmic kinases (RLCKs). The 149 RLCKs of Arabidopsis were divided into 17 subfamilies (RLCK-II and RLCK-IV to RLCK-XIX), based on sequence homology [3]. RLCKs often associate with RLKs to mediate cellular signalling in response to various RLK-sensed environmental and/or developmental signals [4]. Most of the RLCKs, however, have unknown functions.

The Arabidopsis RLCK-VI family is divided into two groups, with seven members each: RLCK VI\_A and RLCK VI\_B [5]. RLCK VI\_A but not RLCK VI\_B kinases were shown to bind plant Rho-type small GTPases (ROPs) in their GTP-bound state [6–9]. This binding results in augmented in vitro kinase activity [6,8,9]. Regulation of kinase activity by Rho-type GTPases is well known in animal and yeast cells as well. In these organisms, the kinase classes regulated by Rho-type Rho/Rac/Cdc42 GTPases are the p21-activated kinases (PAKs), the Rho-kinases (ROKs), the mixed-lineage kinases (MLKs), the myotonin-related Cdc42-binding kinases (MRCKs), the citron kinases (CRIKs), and the novel protein kinase (PKN) [10]. However, plant genomes code for none of these kinases [11]. It seems that, during the evolution of land plants, a sub-group of plant-specific RLCKs were co-opted to mediate ROP GTPase signalling [12]. While the GTPase-binding ability of yeast and animal Rho-type GTPase-regulated kinases is due to the presence of defined structural elements outside of their kinase domains (such as the Cdc42/Rac-interactive binding -CRIB- motif of PAKs), RLCK VI\_A kinases use conserved amino acids widely distributed in the kinase domain to form a binding surface for ROPs [12].

At present, the members of the RLCK VI\_A group are the only known plant kinases for which the activity is directly regulated by ROP GTPases, at least in vitro [6,8,9]. This fact is rather surprising, considering the wide role of animal Rho-type GTPases in kinase signalling [10], as well as the central role of ROP GTPases in a variety of cellular functions [13]. Despite their unique regulation, the biological function of RLCK VI\_A kinases has hardly been investigated so far [11]. The barley HvRBK1 kinase (homologue of the Arabidopsis RLCK VI\_A3 kinase) was shown to have a role in basal disease resistance [6]. Transient silencing of the gene decreased the stability of cortical microtubules and promoted fungal penetration into barley epidermal cells. Mutation in the gene coding for the Arabidopsis homologue of HvRBK1, AtRLCK VI\_A3 was reported to support fungal reproduction [9]. Arabidopsis *AtRBK1* (*RLCK VI\_A4*) and *AtRBK2* (*RLCK VI\_A6*) genes were shown to have augmented expression following pathogen infection, supporting the general role of RLCK VI\_A kinases in pathogen responses. The *atrlck vi\_a3* mutant also exhibited reduced plant size and an increase in the ratio of trichomes with high branch numbers, while the AtRBK1 (RLCK VI\_A4) kinase was found to be a member of a kinase cascade regulating auxin-mediated cell elongation, consistent with a developmental/morphogenic role [14].

Here, we report the involvement of the *AtRLCK VI\_A2* gene in the regulation of plant growth and (skoto)morphogenesis. T-DNA insertion into the gene resulted in reduced hypocotyl elongation and smaller rosette size, which could be ascribed to limited cell expansion. These mutant phenotypes were complemented by the exogenous application of gibberellic acid. Measurements could not reveal differences neither in the gibberellin content nor in the gibberellin sensitivity of the mutant. Transcript analysis indicated that the kinase might indirectly affect gibberellin-dependent responses during skotomorphogensis, by overlapping with the action of the central transcription factor network regulating hypocotyl growth, and interfering with hormone signalling, cell wall organisation, and cellular transport processes. Transcript analysis indicated that the kinase might indirectly affect gibberellin-dependent responses during skotomorphogensis, by overlapping with the action of the central transcription factor network regulating hypocotyl growth, and interfering with hormone signalling, cell wall organisation, and cellular transport processes. **2. Results** 

differences neither in the gibberellin content nor in the gibberellin sensitivity of the mutant.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 20

Here, we report the involvement of the *AtRLCK VI\_A2* gene in the regulation of plant growth and (skoto)morphogenesis. T-DNA insertion into the gene resulted in reduced hypocotyl elongation and smaller rosette size, which could be ascribed to limited cell expansion. These mutant phenotypes

#### **2. Results** *2.1. Molecular Characterization of the RLCK VI\_A2 T-DNA Insertion Mutant and the Transgenic Plants*

#### *2.1. Molecular Characterization of the RLCK VI\_A2 T-DNA Insertion Mutant and the Transgenic Plants Used in the Study Used in the Study*  In order to reveal possible biological functions of the ROP GTP-ase binding AtRLCK VI\_A2

In order to reveal possible biological functions of the ROP GTP-ase binding AtRLCK VI\_A2 (At2G18890) kinase [8], we carried out a search in the GABI-Kat Arabidopsis T-DNA insertional mutant collection [15]. Two lines with predicted T-DNA insertion in the 5' untranslated region (GABI\_676D12) or in the second intron of the At2G18890 gene (GABI\_435H03), respectively, could be identified. Seeds of the T-DNA mutants were obtained from the Nottingham Arabidopsis Stock Centre (NASC) [16]. Expression of the At2G18890 gene was tested in the homozygous lines using a specific PCR primer pair, amplifying the whole transcript by RT-PCR. It could be established that the GABI\_435H03 line does not produce *RLCK VI\_A2* transcripts in contrast to the line GABI\_676D12 (Figure 1a). In order to determine the exact T-DNA insertion site, the junction region of the At2G18890 gene and the T-DNA was amplified from the genomic DNA of the GABI\_435H03 line using a T-DNA-specific reverse primer and an At2G18890 second exon specific forward PCR primer. Sequencing of the PCR product verified that the T-DNA insertion was located not in the second intron but in the third exon (at the ninth codon after the intron/exon junction) (Figure 1b). Mapping transcript reads of the *rlck vi\_a2* mutant by next generation sequencing (NGS) to the reference *Arabidopsis thaliana* genome confirmed that, although the first two exons are transcribed in the mutant, full length functional transcripts are not produced and therefore the mutant can be considered as a knock out (Supplementary Figure S1). (At2G18890) kinase [8], we carried out a search in the GABI-Kat Arabidopsis T-DNA insertional mutant collection [15]. Two lines with predicted T-DNA insertion in the 5' untranslated region (GABI\_676D12) or in the second intron of the At2G18890 gene (GABI\_435H03), respectively, could be identified. Seeds of the T-DNA mutants were obtained from the Nottingham Arabidopsis Stock Centre (NASC) [16]. Expression of the At2G18890 gene was tested in the homozygous lines using a specific PCR primer pair, amplifying the whole transcript by RT-PCR. It could be established that the GABI\_435H03 line does not produce *RLCK VI\_A2* transcripts in contrast to the line GABI\_676D12 (Figure 1a). In order to determine the exact T-DNA insertion site, the junction region of the At2G18890 gene and the T-DNA was amplified from the genomic DNA of the GABI\_435H03 line using a T-DNAspecific reverse primer and an At2G18890 second exon specific forward PCR primer. Sequencing of the PCR product verified that the T-DNA insertion was located not in the second intron but in the third exon (at the ninth codon after the intron/exon junction) (Figure 1b). Mapping transcript reads of the *rlck vi\_a2* mutant by next generation sequencing (NGS) to the reference *Arabidopsis thaliana* genome confirmed that, although the first two exons are transcribed in the mutant, full length functional transcripts are not produced and therefore the mutant can be considered as a knock out (Supplementary Figure S1).

**Figure 1.** Expression of the receptor-like cytoplasmic kinase (*RLCK*) *VI\_A2* gene in various Arabidopsis lines used in the study. (**a**) RT-PCR results using *RLCK VI\_A2* (upper row) and *GAPC-2* specific (lower row) primers in wild type (**1**), T-DNA insertion line GABI\_435H03 (**2**), T-DNA insertion line GABI\_676D12 (**3**), and the GABI\_435H03 line expressing the *RLCK VI\_A2* cDNA transgene under the control of the 35S promoter (complemented mutant) (**4**). (**b**) Site of the T-DNA **Figure 1.** Expression of the receptor-like cytoplasmic kinase (*RLCK*) *VI\_A2* gene in various Arabidopsis lines used in the study. (**a**) RT-PCR results using *RLCK VI\_A2* (upper row) and *GAPC-2* specific (lower row) primers in wild type (**1**), T-DNA insertion line GABI\_435H03 (**2**), T-DNA insertion line GABI\_676D12 (**3**), and the GABI\_435H03 line expressing the *RLCK VI\_A2* cDNA transgene under the control of the 35S promoter (complemented mutant) (**4**). (**b**) Site of the T-DNA insertion in the third exon of the At2G18890 gene coding for the AtRLCK VI\_A2 kinase in the GABI\_435H03 line.

To validate the mutant phenotypes, various transgenic Arabidopsis lines were produced. The *rlck vi\_a2* mutant was complemented with 35S-promoter-driven expression of the At2G18890 cDNA N-terminally fused, with a TAP-tag that allows co-immunoprecipitation of kinase interacting

GABI\_435H03 line.

proteins [17]. Based on gene expression verification, a representative line was selected for further studies (Figure 1a). Furthermore, estradiol-induced RNA-interference [18] was used to knock down *RLCK VI\_A2* expression (Supplementary Figure S2a) to further verify some of the experimental findings obtained with the mutant line. proteins [17]. Based on gene expression verification, a representative line was selected for further studies (Figure 1a). Furthermore, estradiol-induced RNA-interference [18] was used to knock down *RLCK VI\_A2* expression (Supplementary Figure S2a) to further verify some of the experimental findings obtained with the mutant line.

*rlck vi\_a2* mutant was complemented with 35S-promoter-driven expression of the At2G18890 cDNA N-terminally fused, with a TAP-tag that allows co-immunoprecipitation of kinase interacting

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 20

insertion in the third exon of the At2G18890 gene coding for the AtRLCK VI\_A2 kinase in the

#### *2.2. The RLCK VI\_A2 Kinase Controls Seedling and Plant Growth 2.2. The RLCK VI\_A2 Kinase Controls Seedling and Plant Growth*

The *rlck vi\_a2* mutant seedlings that were grown under 8 h/16 h light/dark periods for 5 days exhibited significantly shorter hypocotyls as compared to the wild type (Figure 2a,c). Ectopic expression of the kinase in the mutant background restored hypocotyl growth to normal (Figure 2a,c). The cotyledons were also smaller in the mutant, although this difference was not statistically significant (Figure 2b). Since hypocotyl growth is accelerated in the dark, seeds were also germinated and cultured under continuous darkness for 16 days, and the size of the hypocotyls and cotyledons was compared (Figure 2d–f). The *rlck vi\_a2* mutant seedlings that were grown under 8 h/16 h light/dark periods for 5 days exhibited significantly shorter hypocotyls as compared to the wild type (Figure 2a,c). Ectopic expression of the kinase in the mutant background restored hypocotyl growth to normal (Figure 2a,c). The cotyledons were also smaller in the mutant, although this difference was not statistically significant (Figure 2b). Since hypocotyl growth is accelerated in the dark, seeds were also germinated and cultured under continuous darkness for 16 days, and the size of the hypocotyls and cotyledons was compared (Figure 2d–f).

**Figure 2.** The *rlck vi\_a2* mutation affects hypocotyl and cotyledon elongation. Hypocotyl (**a**,**d**) and cotyledon (**b**,**e**) length were measured for 5-days-old short-day (SD; 8/16h light/dark cycle) and 16 days-old dark-grown (cDark; continuous dark) seedlings. WT—wild type; MUT—T-DNA insertion mutant line; CO—complemented mutant line. Three biological replicates were made with 15–25 **Figure 2.** The *rlck vi\_a2* mutation affects hypocotyl and cotyledon elongation. Hypocotyl (**a**,**d**) and cotyledon (**b**,**e**) length were measured for 5-days-old short-day (SD; 8/16h light/dark cycle) and 16-days-old dark-grown (cDark; continuous dark) seedlings. WT—wild type; MUT—T-DNA insertion mutant line; CO—complemented mutant line. Three biological replicates were made with 15–25 plants per line. Averages and standard errors are shown. Corresponding representative images are displayed on (**c**,**f**). \*\* *p* < 0.005 (Student's *t*-test; comparison to WT).

The hypocotyl of the mutant was found to be significantly shorter under this condition as well, while the wild type and complemented lines exhibited similar hypocotyl sizes (Figure 2d,f). The length of cotyledons, especially that of their petioles, were significantly reduced in the mutant but was restored to the wild type level in the complemented line (Figure 2e,f). The phenotypes of mutant seedlings could be recreated via estradiol-induced silencing of the *RLCK VI\_A2* gene in transgenic seedlings (Supplementary Figure S2b). while the wild type and complemented lines exhibited similar hypocotyl sizes (Figure 2d,f). The length of cotyledons, especially that of their petioles, were significantly reduced in the mutant but was restored to the wild type level in the complemented line (Figure 2e,f). The phenotypes of mutant seedlings could be recreated via estradiol-induced silencing of the *RLCK VI\_A2* gene in transgenic seedlings (Supplementary Figure S2b). Seedlings were also grown into plants in pots in the greenhouse under short-day condition (8 h light, 16 h dark). A significant difference in the size of the rosettes was observed: it was decreased in

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 20

plants per line. Averages and standard errors are shown. Corresponding representative images are

The hypocotyl of the mutant was found to be significantly shorter under this condition as well,

displayed on (**c**,**f**). \*\* *p* < 0.005 (Student's *t*-test; comparison to WT).

Seedlings were also grown into plants in pots in the greenhouse under short-day condition (8 h light, 16 h dark). A significant difference in the size of the rosettes was observed: it was decreased in the mutant, but restored to the normal level in the complemented transgenic line as compared to the wild type control (Figure 3). the mutant, but restored to the normal level in the complemented transgenic line as compared to the wild type control (Figure 3). Altogether, these data indicate that the RLCK VI\_A2 kinase is required for normal plant growth under light as well as in dark; in seedlings as well as in greenhouse plants.

**Figure 3.** Greenhouse-grown mutant (MUT) plants exhibited smaller plant size, as evidenced by measuring the rosette diameter. Normal size of the wild type (WT) plants was restored by expressing the kinase cDNA in the mutant background (complemented, CO line). Rosette diameters in mm are shown in (**a**), and representative images of the measured 4-weeks-old plants in (**b**). The plants were grown in short day conditions (SD). Averages and standard errors were calculated and are shown on (**a**). *n* = 15–25, \*\* *p* < 0.005 (Student's *t*-test; comparison to WT). **Figure 3.** Greenhouse-grown mutant (MUT) plants exhibited smaller plant size, as evidenced by measuring the rosette diameter. Normal size of the wild type (WT) plants was restored by expressing the kinase cDNA in the mutant background (complemented, CO line). Rosette diameters in mm are shown in (**a**), and representative images of the measured 4-weeks-old plants in (**b**). The plants were grown in short day conditions (SD). Averages and standard errors were calculated and are shown on (**a**). *n* = 15–25, \*\* *p* < 0.005 (Student's *t*-test; comparison to WT).

*2.3. The RLCK VI\_A2 Kinase Controls Cell Size*  Altogether, these data indicate that the RLCK VI\_A2 kinase is required for normal plant growth under light as well as in dark; in seedlings as well as in greenhouse plants.

Microscopic investigations revealed that epidermal cell size was significantly smaller in both

#### investigated organs of the mutant seedlings (Figure 4). In the mutant, hypocotyl epidermal cells were *2.3. The RLCK VI\_A2 Kinase Controls Cell Size*

less elongated (Figure 4a,b), while the epidermal cells of the cotyledon were not only smaller in area, but their shape was also different; their circularity index was much higher (Figure 4c,d), indicating limited planar polarity [19]. Microscopic investigations revealed that epidermal cell size was significantly smaller in both investigated organs of the mutant seedlings (Figure 4). In the mutant, hypocotyl epidermal cells were less elongated (Figure 4a,b), while the epidermal cells of the cotyledon were not only smaller in area, but their shape was also different; their circularity index was much higher (Figure 4c,d), indicating limited planar polarity [19].

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 6 of 20

**Figure 4.** The sizes of hypocotyl (**a**,**b**) and cotyledon (**c,d**) cells are significantly smaller in the mutant (MUT) than in the wild type (WT) plants. Fluorescent (**a**) and scanning electron microscopic (**c**) images are shown for the epidermal cells of the hypocotyl (**a**) and the cotyledon (**c**), respectively, of 5-dayold seedlings. The white bars indicate 100 μm (**a**), and 10 μm (**b**), respectively. For the quantitative comparison of cell size (**b**,**d**), 150–200 cells were measured for each of three randomly selected seedlings per line. Averages and standard errors are shown on the histograms. \* *p* < 0.05; \*\* *p* < 0.005 (Student's *t*-test; comparison to WT). **Figure 4.** The sizes of hypocotyl (**a**,**b**) and cotyledon (**c**,**d**) cells are significantly smaller in the mutant (MUT) than in the wild type (WT) plants. Fluorescent (**a**) and scanning electron microscopic (**c**) images are shown for the epidermal cells of the hypocotyl (**a**) and the cotyledon (**c**), respectively, of 5-day-old seedlings. The white bars indicate 100 µm (**a**), and 10 µm (**b**), respectively. For the quantitative comparison of cell size (**b**,**d**), 150–200 cells were measured for each of three randomly selected seedlings per line. Averages and standard errors are shown on the histograms. \* *p* < 0.05; \*\* *p* < 0.005 (Student's *t*-test; comparison to WT). **Figure 4.** The sizes of hypocotyl (**a**,**b**) and cotyledon (**c,d**) cells are significantly smaller in the mutant (MUT) than in the wild type (WT) plants. Fluorescent (**a**) and scanning electron microscopic (**c**) images are shown for the epidermal cells of the hypocotyl (**a**) and the cotyledon (**c**), respectively, of 5-dayold seedlings. The white bars indicate 100 μm (**a**), and 10 μm (**b**), respectively. For the quantitative comparison of cell size (**b**,**d**), 150–200 cells were measured for each of three randomly selected seedlings per line. Averages and standard errors are shown on the histograms. \* *p* < 0.05; \*\* *p* < 0.005

#### *2.4. Gibberellic Acid Treatment Rectifies the rlck vi\_a2 Mutant Phenotypes 2.4. Gibberellic Acid Treatment Rectifies the rlck vi\_a2 Mutant Phenotypes* (Student's *t*-test; comparison to WT).

In order to test whether the mutant phenotype can be linked to the disturbed action of plant hormones, seedlings were grown in the presence of various plant growth regulators (5 nM indoleacetic acid, 1 μM brassinolide, 100 μM ethephon (Ethrel) or 20 μM gibberellic acid (GA3)) and hypocotyl, cotyledon and rosette sizes were measured (Supplementary Figures S3 and S4). Of the investigated hormones, exogenous gibberellin (GA3) was found to rectify the growth defects of the *rlck vi\_a2* mutant (Figure 5) and the RNAi-silenced lines (Supplementary FigureS4). In order to test whether the mutant phenotype can be linked to the disturbed action of plant hormones, seedlings were grown in the presence of various plant growth regulators (5 nM indoleacetic acid, 1 µM brassinolide, 100 µM ethephon (Ethrel) or 20 µM gibberellic acid (GA3)) and hypocotyl, cotyledon and rosette sizes were measured (Supplementary Figures S3 and S4). Of the investigated hormones, exogenous gibberellin (GA3) was found to rectify the growth defects of the *rlck vi\_a2* mutant (Figure 5) and the RNAi-silenced lines (Supplementary Figure S4). *2.4. Gibberellic Acid Treatment Rectifies the rlck vi\_a2 Mutant Phenotypes*  In order to test whether the mutant phenotype can be linked to the disturbed action of plant hormones, seedlings were grown in the presence of various plant growth regulators (5 nM indoleacetic acid, 1 μM brassinolide, 100 μM ethephon (Ethrel) or 20 μM gibberellic acid (GA3)) and hypocotyl, cotyledon and rosette sizes were measured (Supplementary Figures S3 and S4). Of the investigated hormones, exogenous gibberellin (GA3) was found to rectify the growth defects of the *rlck vi\_a2* mutant (Figure 5) and the RNAi-silenced lines (Supplementary FigureS4).

**Figure 5.** Exogenous gibberellin treatments complemented the mutant phenotypes. Wild type (WT) or *rlck vi\_a2* mutant (MUT) seedlings grown in vitro in short days (SD; 8 h/16 h light/dark) for 6 days (**a**)

or in continuous dark (cDark) for 17 days (**b**,**c**) and plants grown at short days in greenhouse (**d**) were or were not treated with 20 µM gibberellic acid (GA<sup>3</sup> ). Hypocotyl length (**a**,**b**), cotyledon length (**c**) or rosette diameter (**d**) were measured in 15–25 seedlings or plants, respectively, in three repetitions. Averages and standard errors are shown. \*\* *p* < 0.05 (Student's *t*-test; comparison to WT). (**a**) or in continuous dark (cDark) for 17 days (**b**,**c**) and plants grown at short days in greenhouse (**d**) were or were not treated with 20 μM gibberellic acid (GA3). Hypocotyl length (**a**,**b**), cotyledon length (**c**) or rosette diameter (**d**) were measured in 15–25 seedlings or plants, respectively, in three repetitions. Averages and standard errors are shown. \*\* *p* < 0.05 (Student's *t*-test; comparison to WT).

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 7 of 20

or *rlck vi\_a2* mutant (MUT) seedlings grown in vitro in short days (SD; 8 h/16 h light/dark) for 6 days

#### *2.5. The E*ff*ect of the rlck vi\_a2 Mutation on Gibberellic Acid Level, Synthesis, and Signalling in Seedlings 2.5. The Effect of the rlck vi\_a2 Mutation on Gibberellic Acid Level, Synthesis, and Signalling in Seedlings*

Endogenous level of gibberellins (GAs) having biological activity was determined in the case of wild type, and mutant and complemented mutant seedlings (Figure 6a). The major bioactive GAs include GA1, GA3, GA4, and GA7, but GA<sup>5</sup> and GA<sup>6</sup> have also been indicated to have biological activities [20]. In 9-day-old seedlings, among these GAs, GA<sup>5</sup> exhibited the highest concentration, GA1, GA<sup>4</sup> and GA<sup>7</sup> was found to have lower levels, while GA<sup>3</sup> and GA<sup>6</sup> could not be detected. No significant differences were found in the concentration of active GAs in the seedlings of the various Arabidopsis lines tested. The GA sensitivity of the mutant was also compared to that of the wild type control. It was found that hypocotyl growth was not less responsive to exogenous GA<sup>3</sup> in the mutant than in the wild type (Figure 6b). Endogenous level of gibberellins (GAs) having biological activity was determined in the case of wild type, and mutant and complemented mutant seedlings (Figure 6a). The major bioactive GAs include GA1, GA3, GA4, and GA7, but GA5 and GA6 have also been indicated to have biological activities [20]. In 9-day-old seedlings, among these GAs, GA5 exhibited the highest concentration, GA1, GA4 and GA7 was found to have lower levels, while GA3 and GA6 could not be detected. No significant differences were found in the concentration of active GAs in the seedlings of the various Arabidopsis lines tested. The GA sensitivity of the mutant was also compared to that of the wild type control. It was found that hypocotyl growth was not less responsive to exogenous GA3 in the mutant than in the wild type (Figure 6b).

**Figure 6.** Gibberellin content and gibberellin sensitivity of the mutant and the wild type. (**a**) Endogenous content of active gibberellins was measured in 9-days-old seedlings of wild type (WT), *rlck vi\_a2* mutant (MUT), and complemented mutant (CO) lines. Seedlings were grown in vitro under short day (SD; 8 h/16 h light/dark) conditions in a growth chamber. Samples were collected from three independent experiments. Averaged data are shown with the standard deviations. No statistically significant differences could be observed among the tested Arabidopsis lines (*p* < 0.05 Student's *t*-test; comparison to WT). (**b**) Relative hypocotyl length was determined in response to a range of GA3 concentrations (0–100 μM), in the case of wild type and mutant seedlings (9-days-old; grown under low intensity continuous white light). **Figure 6.** Gibberellin content and gibberellin sensitivity of the mutant and the wild type. (**a**) Endogenous content of active gibberellins was measured in 9-days-old seedlings of wild type (WT), *rlck vi\_a2* mutant (MUT), and complemented mutant (CO) lines. Seedlings were grown in vitro under short day (SD; 8 h/16 h light/dark) conditions in a growth chamber. Samples were collected from three independent experiments. Averaged data are shown with the standard deviations. No statistically significant differences could be observed among the tested Arabidopsis lines (*p* < 0.05 Student's *t*-test; comparison to WT). (**b**) Relative hypocotyl length was determined in response to a range of GA<sup>3</sup> concentrations (0–100 µM), in the case of wild type and mutant seedlings (9-days-old; grown under low intensity continuous white light).
