Selection of StGI.04-repressed lines
A 250-bp fragment of St.GI04 with the less, but still 71.2% identity with the corresponding fragment of StGI.12 (Additional file 1: Fig. S1), was used for generation of StGI.04-repressed ‘Désirée’ (DES) lines (aGI lines). Leaves harvested from plants grown in vitro were tested for the level of repression using reverse-transcription polymerase chain reaction (RT-PCR). Twenty lines were found with lower level of StGI.04 expression than the non-transformed control DES out of which five lines (aGI43, aGI44, aGI52, aGI53, aGI55) with different levels of reduction in StGI.04 transcript level were selected for further studies. Expression of StGI.04 was quantified in the selected five lines with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The highest repression, 51% of StGI.04 mRNA detected in DES, was found in aGI52, while only a minimal repression, a 2% reduction, was found in aGI55 (Fig. 1a).
The five selected lines were propagated in vitro, transferred to pots, and grown further under greenhouse conditions in 12 parallel setups. Expression of StGI.04 was re-tested in leaves (Fig. 1b). Four lines possessed significantly (p < 0.01) lower StGI.04 expression than found in DES. As in leaves of in vitro plants, a smaller difference compared to DES was found in aGI55, but even this difference was significant at the p < 0.05 level. The StGI.04 transcript level was a little bit higher, 63% versus 51%, in aGI52 leaves of greenhouse-grown plants versus leaves of in vitro-grown plants.
At the end of the vegetation period, the tubers were harvested, and the level of StGI.04 expression was tested in tubers (Fig. 1c). In line with the lowest expression in leaves (Fig. 1b), the lowest expression was detected in tubers of the aGI43 plants (Fig. 1c). All lines, except aGI44, showed a significant (p < 0.01) level of StGI.04 repression in tubers, including aGI52 with 67% of wild type StGI.04 mRNA level.
Specificity of repression in aGI52
The line aGI52 showed a significant (p < 0.01) and relatively stable level of reduction in StGI.04 expression based on all three RT-qPCR analyses (Fig. 1). Therefore, this line was selected for further detailed studies.
Although the region of reduced sequence similarity between StGI.04 and StGI.12 was used for generation of aGI lines, the identity of the two regions was still substantial. Thus, the repression of StGI.12 expression by the StGI.04 fragment could not be excluded. To test this possibility, an StGI.12-specific primer pair [8] was used in parallel with the StGI.04-specific primer pair [8] in the RT-qPCR analysis of aGI52 and DES leaves and tubers. Figure 2 shows that the repression in aGI52 was StGI.04-specific and did not extend to StGI.12.
Phenotypes and tuberisation of aGI plants
Development and morphology of aGI43, aGI44, aGI52, aGI53, and aGI55 plants grown in the greenhouse were visually followed and compared to DES. Plant heights were measured at 7 weeks after transferring them from in vitro into pots. Neither phenotypic changes nor height differences were observed. The earliness of tuberisation was tested also at 7 weeks after planting by counting the number of tubers after carefully tipping the plants out of the pots. Significant delay in tuber initiation was detected only in the line aGI44. After counting, the plants were replaced in the pots and grown until the end of the vegetation period when the tubers were harvested and measured for weight. No differences in tuber yield were obtained between the aGI lines and DES. The size distribution of aGI tubers was also similar to DES, peaking at 8 to 10 cm in diameter, except aGI44 and aGI55, both of which produced a larger number of small tubers than DES (Additional file 1: Fig. S2).
The molecular model of tuber formation is based on S. andigena, a strict SD plant for tuberisation [7]. Therefore, we wanted to test the effects of StGI.04 repression not only under 12 h light/12 h dark (LD conditions) but also under SD conditions (8 h light/16 h dark). The aGI52 line was compared to DES in this experiment. Even under SD conditions, no difference in canopy phenotype or tuber yield was detected between aGI52 and DES (Additional file 1: Fig. S2).
Anthocyanin content of tuber peels
DES is a red-skinned potato. The skin colour of aGI tubers collected from greenhouse-grown plants was lighter than the skin colour of DES (Additional file 1: Fig. S3a) although, to different extents. The reduction in colour was the most pronounced in aGI52, aGI53 and aGI44. The difference in tuber skin colour was also obvious between aGI52 and DES grown under SD conditions (Fig. 3a). Since anthocyanins determine the skin colour [9], these compounds were extracted from tuber peels and their relative quantity measured. When compared with DES, a 52, 36 and 31% reduction in anthocyanin content was found in greenhouse-grown aGI53, aGI44 and aGI52 tuber peels, respectively (Additional file 1: Fig. S3b). A similar, 43% reduction was observed in the anthocyanin content of aGI52 tuber skins developed under SD conditions (Fig. 3b).
High-performance liquid chromatography (HPLC) was used to specify the anthocyanins extracted from tuber peels. Three anthocyanins, cyanidin 3,5-di-O-glucoside, pelargonidin 3,5-di-O-glucoside and delphinidin 3,5-di-glycoside were detected with pelargonidin 3,5-di-O-glycoside being present in the largest amount. A significant reduction in cyanidin 3,5-di-O-glucoside and pelargonidin 3,5-di-O-glycoside content of aGI52 peels compared to DES peels was demonstrated (Fig. 3c). An attempt was also made to detect malvinidin 3-galactoside; however, it was not present in a detectable amount.
Transcriptome analysis of aGI52 leaves
To explore the effects of StGI.04 repression on the global transcription profile, the same leaf RNA of greenhouse-grown aGI52 and DES plants tested for the specificity of repression (Fig. 2) was used for RNA-seq analysis in three biological replicates. Parameters presented in Additional files Table S1, Fig. S4 and S5 indicate that the analysis had a good quality.
To assess the alterations in unigene expression we analysed the differentially expressed unigenes (|log2 (Fold Change)| > 1 and padj < 0.05) in the comparison of aGI52 and DES. We found 454 and 247 uniquely expressed genes in DES and aGI52, respectively (Fig. 4a). We obtained 488 differentially expressed genes (DEGs): (1) 289 were up- and (2) 199 were down-regulated in aGI52 (Fig. 4b and Additional file 1: Fig. S6). Gene ontology (GO) enrichment analysis revealed that mainly those genes were up-regulated, which are related to photosynthesis, while peptidase regulators and inhibitors were down-regulated in aGI52 (Fig. 4c, d and Additional file 1: Fig. S7). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the glyoxylate and dicarboxylate metabolism, the carbon metabolism and the peroxisomal pathways were activated, while the metabolisms of some amino acids were suppressed (Fig. 4e). Nevertheless, none of these pathways were significantly altered (corrected p > 0.05).
Transcription factors (TFs) are key regulators of plant development and stress responses. Thus, first we focused on differentially expressed TFs. Fourteen up-regulated and 11 down-regulated TFs were identified in aGI52 (Fig. 5a). The up-regulated category included the gene for bHLH63/CIB1, a positive regulator of flower development [11], NF-YB-3/HAP3C, which can interact with CO when it replaces HAP2 in the ternary HAP complex and promotes flowering in Arabidopsis [12], IBH1 that negatively regulates cell and organ elongation in response to gibberellin and brassinosteroid signalling [13], an ABI5-like gene similar to the major mediator of ABA repression of growth and floral transition in Arabidopsis [14, 15] and RVE1, a clock output affecting plant development [16]. Furthermore, TF genes involved in plant defence responses, ERF1B, ZAT10, WRKY11, MYB1R1, and TGA2.1, were also activated [17,18,19,20,21] in addition to ASR3 that acts in an opposite and negative manner to regulate immune gene expression in Arabidopsis [22]. Interestingly, unlike NF-YB-3/HAP3C, NF-YA-1/HAP2A, encoding the DNA-binding subunit of the HAP complex, is down-regulated. RAP2–7 encoding an ethylene responsive TF with an APETALA2 (AP2) domain is also down-regulated. AP2-like target genes act as floral repressors [23]. Furthermore, GATA21, a repressor of the gibberellin signalling pathway that also represses flowering [24], and a CONSTANS-LIKE gene, COL13, is expressed at lower levels in aGI52 than in DES. Figure 5b demonstrates that the expression of the majority of TFs listed in Fig. 5c are highly synchronised. The exceptions are ZAT10, ERF1B, and bHLH72, whose expression pattern is similar only to that of TGA2.1, MYB1R-1, and ASR3.
The KEGG analysis highlighted some DEGs involved in glyoxylate and dicarboxylate metabolism, carbon metabolism, and peroxisomal pathways (Additional file 1: Fig. S8–10). These included the genes encoding the key enzymes of starch synthesis, ADP-GLUCOSE SYNTHASE (AGS), STARCH SYNTHASE (SS), and STARCH PHOSPHORYLASE (SP) in addition to TREHALOSE-6-PHOSPHATE SYNTHASE (TPS), a sugar messenger connecting metabolism and development, and the first line defence antioxidants, SUPEROXIDE DISMUTASE (SOD) and CATALASE (CAT) as shown in Fig. 6a. Nevertheless, while SOD was up-regulated, CAT was down-regulated (Fig. 6b). In contrast, down-regulation of starch synthesis genes was highly coordinated (Fig. 6c).
Although the anthocyinin content in leaves was so low that only cyanidin 3,5-di-O-glucoside could be detected the difference in the concentration of this compound between aGI52 (22.4 ± 0.2 ng/g Fw) and DES (24.1 ± 0.2 ng/g Fw) was significant (p < 0.01). This prompted us to make a manual search for DEGs involved in anthocyanin metabolism. Anthocyanins are a class of flavanoids synthesized through a branch of the phenylpropanoid pathway. The beginning genes include PHENYLALANINE AMMONIA LYASE (PAL), CINNAMATE 4-HYDROXYLASE (C4H), and 4-COUMARYOL COA LIGASE (4CL). The next steps are divided into early and late enzymatic steps. The early biosynthetic genes include CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), FLAVANONE 3-HYDROXYLASE (F3H), and FLAVANONE 3′-HYDROXYLASE (F3’H). The late biosynthesis genes include DIHYDROFLAVONOL 4-REDUCTASE (DFR), ANTHOCYANIDIN SYNTHASE (ANS), and UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYL TRANSFERASE (UFGT). A correlation between the expression of these genes and anthocyanin content of potato tubers was found. The pathway is transcriptionally regulated by the MBW complex consisting of MYB, bHLH, and WD40 TFs (reviewed in [25]). Nevertheless, none of the coding genes of the above listed proteins except PAL (log2 fold-change − 0.5) was differentially expressed in aGI52 leaves compared to DES leaves. Anthocyanin discoloration might be due to either lower level of synthesis or higher level of degradation. For anthocyanin degradation, the candidate gene families are POLYPHENOL OXIDASES, PEROXIDASES, and β-GLUCOSIDASES (reviewed in [25]). We found no change in expression of any of these genes in aGI52 although we found the MYB-RELATED PROTEIN Hv1 (log2 fold-change 0.87) among the up-regulated TF genes (MYB-Hv1 in Fig. 5) that may be involved in the regulation of flavonoid biosynthesis and LDOX (log2 fold-change − 0.02), the coding gene of the enzyme oxidising leucoanthocyanidins into anthocyanidins, which was slightly, but significantly down-regulated in aGI52 leaves.
Validation of RNA-seq data by real-time quantitative PCR
To validate the results of RNA-seq data, we selected three significantly up-, and five down-regulated DEGs in aGI52. These genes included four TFs, a diagnostic indicator of sulphur nutritional status, an F-box/LRR-repeat protein, a transmembrane transporter, and a heat shock protein (Fig. 7). The expression trends of each selected gene using RT-qPCR and RNA-seq were similar, indicating that the transcriptome data are highly reliable.
Phylogenetic and similarity analysis of StMYB-Hv1
MYB TFs are one of the largest TF families in plants which contain 1–4 tandem incomplete repeats (Rs) at the N-terminal region. According to the number of Rs the MYB family is divided into four categories (reviewed in [26]). It was found that StMYB-Hv1 is made up of 122 amino acids and belongs to the R3 MYB TFs carrying a single R3 type domain. The phylogenetic analysis showed that StMYB-Hv1 has the highest relationship with the MYB3-like proteins of Nicotiana tomentosiformis and Solanum lycopersicum (Fig. 8a and Additional file 1: Fig. S11). However, since the function of these proteins has not been known thus far a search for the most similar Arabidopsis thaliana protein sequence was carried out. This search resulted in identification of AtMYBL2 as the protein with the highest similarity to StMYB-Hv1 (Fig. 8b).