*2.6. Transcriptome Analysis Indicated a Gibberellin-Independent Role for the RLCK VI\_A2 Kinase in Skotomorphogenesis*

To clarify the role of the RLCK VI\_A2 kinase in seedling growth and cell elongation, the transcriptome of 18-days-old dark-grown wild-type and *rlck vi\_a2* mutant seedlings were compared in three biological replicates. 1203 statistically highly significant (*q* < 0.05; fold change > 1.5) DEGs (differentially expressed genes) were identified, among which 406 were upregulated and 707 downregulated (Supplementary Table S1). Gene ontology (GO) enrichment analysis (Plant GOSlim ontologies) indicated the involvement of the kinase in the responses to various exogenous and endogenous stimuli including biotic and abiotic stresses and plant hormones as well as in lipid, carbohydrate, and secondary metabolism (Table 1). A more detailed GO analysis (complete GO ontologies) showed that the kinase might modulate the signalling, metabolism and transport of several hormones, including auxin, abscisic acid, jasmonic acid, and salicylic acid, but gibberellic acid-related DEGs were not significantly enriched in the mutant (Supplementary Tables S2 and S3).


**Table 1.** Gene ontology classification (Plant GOSlim) of the DEGs for 17-days-old dark-grown *rlck vi\_a2* mutant seedlings without roots in comparison to wild type.


**Table 1.** *Cont.*

FDR—false discovery rate; P—biological process; F—molecular function; C—cellular compartment.

Browsing the annotation of those DEGs that have the GO "hormone response" for the key word "gibberellin" revealed a small number of GA-regulated proteins (Table 2 and Supplementary Table S3), including the GAI DELLA-type transcriptional regulator, which exhibited an app. 1,5-fold increased expression in the mutant. The expression of other DELLA protein genes showed a slightly but not statistically significant increase in this line. GA-related DEGs also included the AtHB23 homeobox protein that is involved in light-regulated hypocotyl growth and cotyledon expansion [21] and the GA catabolic enzyme GA2ox6 [22] (Table 2 and Supplementary Table S3). GA transport is considered to be mediated by NPF (NRT1/PTR FAMILY) transporters [23–25]. The NPF family has 53 members in Arabidopsis [26], but the expression of only 6 of them was altered in the *rlck vi\_a2* mutant. Interestingly, four out of these 6 NPFs have already been implicated in GA transport (Table 2 and Supplementary Table S3) [24,25,27]. Expressions of selected GA-related transcripts in the mutant and wild type background were also tested by qRT-PCR (Supplementary Figure S5a). The results supported the validity of the transcriptomic data.




**Table 2.** *Cont.*

\* Manually selected based on the presence of the word "gibberellin" in the annotations of DEGs implicated in "hormone response" by the AgriGO v2.0 tool. \*\* NPF transporters that were reported to transport gibberellins by a [24]; <sup>b</sup> [27]; <sup>c</sup> [25]. \*\*\* Note that only the differential expression of *GAI* was statistically significant. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 10 of 20

Although only a few of them were directly GA-related, the *RLCK VI\_A2*-dependent DEGs

Although only a few of them were directly GA-related, the *RLCK VI\_A2*-dependent DEGs showed an overlap (15%), with the DEGs identified in the GA synthesis defective mutant *ga1-3* [28,29] (Figure 7 and Supplementary Table S4) suggesting an indirect and limited effect of the kinase on GA signalling. showed an overlap (15%), with the DEGs identified in the GA synthesis defective mutant *ga1-3* [28,29] (Figure 7 and Supplementary Table S4) suggesting an indirect and limited effect of the kinase on GA signalling.

Hypocotyl elongation is coordinated by the light and GA-regulated PIF transcription factors [30], as well as by the brassinosteroid- and auxin-controlled transcription factors BZR1 and ARF6, respectively [31]. The transcription factors were shown to interact with each other, forming a central growth regulatory circuit [31]. Promoters of thousands of genes were identified to be direct and partly common targets of the PIF4, BZR1 and ARF6 factors in relation to hypocotyl cell elongation [31]. Among the 1019 DEGs affected by the *rlck vi\_a2* mutation, 317 (31%) belong to direct PIF4 targets, 359 (35%) is the direct target of BZR1 and 176 (17%) of ARF6, respectively, and 100 (9.8%) of them bind all three TFs (Figure 7 and Supplementary Table S5). PIF4 itself was found to be slightly upregulated (1.5-fold) in the mutant. Expressions of selected transcripts involved in the regulation of hypocotyl elongation during skotomorphogenesis were also tested in the mutant and the wild type by qRT-PCR (Supplementary Figure S5b). The obtained results supported the validity of the transcriptomic data. Hypocotyl elongation is coordinated by the light and GA-regulated PIF transcription factors [30], as well as by the brassinosteroid- and auxin-controlled transcription factors BZR1 and ARF6, respectively [31]. The transcription factors were shown to interact with each other, forming a central growth regulatory circuit [31]. Promoters of thousands of genes were identified to be direct and partly common targets of the PIF4, BZR1 and ARF6 factors in relation to hypocotyl cell elongation [31]. Among the 1019 DEGs affected by the *rlck vi\_a2* mutation, 317 (31%) belong to direct PIF4 targets, 359 (35%) is the direct target of BZR1 and 176 (17%) of ARF6, respectively, and 100 (9.8%) of them bind all three TFs (Figure 7 and Supplementary Table S5). PIF4 itself was found to be slightly upregulated (1.5-fold) in the mutant. Expressions of selected transcripts involved in the regulation of hypocotyl elongation during skotomorphogenesis were also tested in the mutant and the wild type by qRT-PCR (Supplementary Figure S5b). The obtained results supported the validity of the transcriptomic data. Altogether, these data imply that GA level/signalling is not the primary target of the RLCK VI\_A2 kinase, at least not at the gene transcription level.

**Figure 7.** Overlaps of the DEGs of the *rlck vi\_a2* mutant with the DEGs of the *ga1-3* gibberellin synthesis mutant [28,29] (**a**) and with those genes for which the promoters are direct targets of the cell/hypocotyl elongation regulatory transcription factors PIF4, BZR1, and/or ARF6 [31] (**b**). **Figure 7.** Overlaps of the DEGs of the *rlck vi\_a2* mutant with the DEGs of the *ga1-3* gibberellin synthesis mutant [28,29] (**a**) and with those genes for which the promoters are direct targets of the cell/hypocotyl elongation regulatory transcription factors PIF4, BZR1, and/or ARF6 [31] (**b**).

Altogether, these data imply that GA level/signalling is not the primary target of the RLCK VI\_A2 kinase, at least not at the gene transcription level.
