Strigolactones (SLs) are a class of phytohormones controlling shoot branching. In potato (Solanum tuberosum), tubers develop from underground stolons, diageotropic stems which originate from basal stem nodes. As the degree of stolon branching influences the number and size distribution of tubers, it was considered timely to investigate the effects of SL production on potato development and tuber life cycle.
Transgenic potato plants were generated in which the CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) gene, key in the SL biosynthetic pathway, was silenced by RNA interference (RNAi).
The resulting CCD8-RNAi potato plants showed significantly more lateral and main branches than control plants, reduced stolon formation, together with a dwarfing phenotype and a lack of flowering in the most severely affected lines. New tubers were formed from sessile buds of the mother tubers. The apical buds of newly formed transgenic tubers grew out as shoots when exposed to light. In addition, we found that CCD8 transcript levels were rapidly downregulated in tuber buds by the application of sprout-inducing treatments.
These results suggest that SLs could have an effect, solely or in combination with other phytohormones, in the morphology of potato plants and also in controlling stolon development and maintaining tuber dormancy.
The control of shoot branching and its impact on plant architecture have fundamental implications for the development of more productive crops (reviewed in Xie et al., 2010; Brewer et al., 2013). Considerable progress has been made in understanding the hormonal control of axillary bud outgrowth. The discovery of the importance of the shoot apex, in decapitation experiments, led to the formulation of the apical dominance theory. According to this, auxin, known then as ‘the growth substance’, inhibits the outgrowth of lateral buds (Thimann & Skoog, 1933). Several early studies focused on the antagonistic roles of auxins and cytokinins (CKs) in controlling bud outgrowth (Skoog & Thimann, 1940; Gunckel & Thimann, 1949; Thimann et al., 1971; Morris, 1977), also detailed in more recent work (Bangerth et al., 2000; Pernisova et al., 2009; Shimizu-Sato et al., 2009).
However, experiments studying long-distance signals in mutants that exhibited increased branching or tillering suggested that factors in addition to auxins and CKs might also be involved in controlling bud outgrowth. Examples include the Arabidopsis thaliana (Arabidopsis) more axillary growth (max), pea (Pisum sativum) ramosus (rms), petunia (Petunia hybrida) decreased apical dominance (dad) and rice (Oryza sativa) high tillering dwarf (htd) mutants (Beveridge et al., 1994; Napoli, 1996; Morris et al., 2001; Stirnberg et al., 2002; Sorefan et al., 2003; Zou et al., 2005; Simons et al., 2007). The study of these mutants led to the identification of strigolactones (SLs) as carotenoid-derived plant hormones with a major role in the determination of axillary bud outgrowth (Gomez-Roldan et al., 2008; Umehara et al., 2008; reviewed by Gong et al., 2012).
SLs are compounds long known for their role as germination stimulants (Cook et al., 1966, 1972) and pre-symbiotic branching factors for arbuscular mycorrhiza (Akiyama et al., 2005). SLs are derived from carotenoids (Matusova et al., 2005) through oxidative cleavage catalysed by carotenoid cleavage dioxygenases (CCDs; Gomez-Roldan et al., 2008; Umehara et al., 2008). Currently, it is thought that CCD7 catalyses the 9,10 cleavage of 9-cis-β-carotene to produce 10′-apo-β-carotenal and β-ionone. Then, the 10′-apo-β-carotenal is cleaved by CCD8 to produce C18-ketone β-apo-13-carotenone. This compound is immediately converted by the same enzyme (CCD8) to carlactone, supposedly involving a series of different reactions, including cis–trans isomerization, Baeyer–Villiger-like rearrangements and repeated dioxygenations (Alder et al., 2012). Carlactone presumably serves as substrate for P450 enzymes (e.g. Arabidopsis MAX1) which catalyse the production of the different forms of SL found in nature.
The role of SLs in plant development has been extended recently from model systems to crop and horticultural plants, including tomato (Vogel et al., 2010; Kohlen et al., 2012), kiwi fruit (Actinidia chinensis; Ledger et al., 2010), chrysanthemum (Dendranthema grandiflorum; Liang et al., 2010) and, recently, maize (Zea mays; Guan et al., 2012). In each of these studies, the down-regulation of CCD7 and/or CCD8 resulted in above-ground phenotypes with increased stem branching.
Potato tubers develop from underground rhizomes or stolons. Usually originating from basal stem nodes, stolons are a type of diageotropic shoot or stem with strongly elongated internodes (Struik, 2007). The number and size distribution of tubers are traits of critical economic importance, strongly influenced by the degree of stolon branching (Celis-Gamboa et al., 2003). In addition, the activation status of tuber apical and axillary buds impacts on important traits, such as tuberization, tuber dormancy and tuber sprout number. These aspects of tuber development are orchestrated by a complex interplay of phytohormone and sugar signals, in which the role of SLs has just started to be investigated in more detail. Thus, a recent study into the regulation of stolon axillary bud growth in potato found evidence that an increase in auxin is associated with tuber formation (Roumeliotis et al., 2012). In an in vitro tuberization system, basally applied auxin can stimulate this process, whereas SL inhibits tuberization (Roumeliotis et al., 2012). It has been demonstrated recently that potato tubers exhibit apical dominance behaviour that is very similar to that of other stems (Teper-Bamnolker et al., 2012). Therefore, in view of the importance of underground stem branching in potato and the potential impact on the tuber life cycle, it was considered timely to investigate the effects of silencing expression of the potato CCD8 gene. The effects on plant phenotype, particularly related to the tuber life cycle, are described in this study.
Materials and Methods
Generation of StCCD8-RNAi construct
Forward (5′-ACCGAGAATTTTCAGAAG-3′) and reverse (5′-TGATGAAGTGGAATGTCACG-3′) primers were used to amplify a 530-bp coding sequence of StCCD8 from stem and root tissue cDNA of Solanum tuberosum L. cv Desiree. This was performed using the Platinum Pfx DNA Polymerase from Invitrogen (UK), following the manufacturer's instructions. The sequence was subcloned into a pDONR201 vector and then cloned into the Ti vector pHellsgate8, under the CaMV 35S promoter, via Gateway technology, according to the manufacturer's instructions (Invitrogen).
Phylogenetic analysis of CCD8 orthologues
Amino acid sequences of CCD8 orthologues, in FASTA format, were aligned using the default settings in MAFFT software (Katoh et al., 2002; http://mafft.cbrc.jp/alignment/server/). Next, the maximum likelihood phylogeny was generated using TOPALi (www.topali.org) under the WAG+G model with the PhyML method. The support for the maximum likelihood phylogenies came from 100 bootstrap re-samplings.
Agrobacterium-mediated transformation of potato plants
The binary vector containing the StCCD8 gene fragment (Supporting Information Fig. S1) was transformed into the Agrobacterium tumefaciens strain LBA4404 by electroporation and selected on kanamycin-containing (100 μg ml−1) and rifampicin-containing (100 μg ml−1) agar media. Six-week-old Solanum tuberosum cv Desiree plants were transformed and cultivated as described by Ducreux et al. (2005).
Growth of plant material
Following Agrobacterium-mediated transformation, putative transgenic plants were grown in a glasshouse at a constant temperature of 20°C (daytime) and 15°C (night-time). The mean day length was 16 h and the light intensity (photosynthetic photon flux density) was between 400 and 1000 μmol m−2 s−1.
Total RNA extraction from potato tissues
For expression analysis, root, stem and tuber tissues were harvested from three plants from each transgenic line or control. Unpooled tissue from these three biological replicates was freeze–dried and total RNA extracted as detailed in Ducreux et al. (2005). The method was effective for all tissues tested, including tubers, stems, leaves and flowers, providing good quality RNA as assessed by gel electrophoresis.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using the Universal Probe Library (UPL)
Invitrogen Superscript™ III reverse transcriptase (www.invitrogen.com) was used for the reverse transcription of 10 μg of total RNA, primed with random hexamers. For qRT-PCR, using the UPL System (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp), 25 ng of cDNA was used as template. The total volume of each reaction was 25 μl, containing gene-specific primers and probe, at a concentration of 0.2 and 0.1 μM, respectively, together with 1× FastStart TaqMan® Probe Master (supplemented with ROX reference dye; Roche Diagnostics Ltd, W. Sussex, UK). The following thermal cycling conditions were used: 10 min of denaturation at 95°C, followed by 40 cycles (15 s at 94°C, 60 s at 60°C). All reactions were carried out in triplicate, using unpooled cDNAs from three biological replicates. For quantification, the relative expression levels were calculated using the internal reference control primers of the Elongation factor-1alpha (EF1α) gene (Nicot et al., 2005), employing the Pfaffl method (Pfaffl, 2001). UPL primer and probe sequences were as follows: StEF1alpha_fd (5′-CTTGACGCTCTTGACCAGATT-3′), StEF1alpha_rev (5′-GAAGACGGAGGGGTTTGTCT-3′), UPL probe number 117 (5′-AGCCCAAG-3′); StCCD8_UPL_fd (5′-AGCATTTGTGCATGTTATGTGTAA-3′), StCCD8_UPL_rev (5′-GGCACTTCTACACTTGCAACAA-3′), UPL probe number 59 (5′-CAGTGGCA-3′); StTCP_UPL_fd (5′-CACCCTAGCTCTTCTTTTACGG-3′), StTCP_UPL_rev (5′-ACACTACTCTTGTTGTTGTTATCGTTC-3′), UPL probe number 131 (5′-CTGGTGGT-3′).
Procedures for the extraction and subsequent measurement of leaf chlorophyll levels were based on the method of Barnes et al. (1992). To ensure the collection of tissue of similar age and experiencing similar environmental conditions, leaf discs (16.5 mm in diameter) were excised from potato leaves sampled from the third node from the apex of four biological replicate potato plants, avoiding the midrib. The discs were then weighed and a measurement of relative chlorophyll was made, as described here. Using a scalpel blade, the leaf discs were cut into small pieces and inserted into a 7-ml brown vial with 5 ml of dimethylsulfoxide (DMSO), followed by incubation in the dark at room temperature for 24 h. The supernatant was transferred into a cuvette for spectrophotometric quantification. Three technical replicates of the absorbance were taken from each of the four biological replicates, measuring two absorbance values for each sample: A649 nm and A665 nm.
Sprout release assay
Discs containing a tuber bud were isolated from mature tubers stored in the cold for 2–3 wk using a size 2 corkborer (6.5 mm in diameter), following the protocol detailed by Hartmann et al. (2011). The tuber discs were incubated for 5 min in 50-μM solutions of 6-benzylaminopurine (BAP) and gibberellic acid (GA3). A solution of 2.5 μM GR24, a synthetic SL purchased from http://www.chiralix.com, dissolved in acetone was prepared by diluting GR24 in 4% PEG 1450, 25% ethanol and 0.05% acetone, and applied in 5-μl volumes to the tuber discs. The sprouting behaviour of the tuber discs was scored daily for 7 d.
GR24 application to potato nodal cuttings
Potato material, previously grown in tissue culture for 4 wk, was excised into single nodes (10–15 per plantlet). These nodes were transferred to 90-mm Petri dishes, with the axillary bud exposed, on Murashige and Skoog medium (Murashige & Skoog, 1962) supplemented with 0.05 mg l−l biotin, 0.5 mg l−l folic acid, pyridoxine-HCl, thiamine-HCl, 2 mg l−l glycine, 100 mg l−l myo-inositol, 5 mg l−l nicotinic acid, 10 mg l−l kinetin, 0.1 mg l−l IAA, 8% (w/v) sucrose, 0.6% (w/v) bacto microagar (Difco; Voigt Global Distribution Inc., Lawrence, USA), pH 5.7 and, where included, 1–10 mg l−1 GR24. After sealing with Nescofilm, the dishes were placed in a 20–25°C incubator in total darkness. Tuberization began in c. 1 wk and microtuber growth was observed for c. 9 wk.
Analysis of carotenoids
Whole-tuber samples were freeze–dried in liquid nitrogen and stored at −80°C before analysis. The extraction of total potato tuber carotenoids and subsequent analysis by reverse-phase high-performance liquid chromatography (HPLC) were carried out as detailed in Morris et al. (2004).
Analysis of SL content in potato root tissue
For the extraction of SLs from root tissues, potato plants were grown under normal conditions and phosphate starvation, making use of an aeroponics system from http://nutriculture.com/index.htm, as described by Liu et al. (2011) for Medicago truncatula. Once harvested, tissue was frozen in liquid nitrogen and freeze–dried. The procedure of extraction was carried out as detailed by Kretzschmar et al. (2012). The combined dried fractions were resuspended in 200 μl of acetonitrile : water (25 : 75, v/v) and filtered through Minisart SRP4 0.45-μm filters (Sartorius, Goettingen, Germany), before analysis by HPLC-MS-MS, as in Kohlen et al. (2012).
Identification of the potato CCD8 gene
A sequence similarity search of the DFCI Potato Gene Index (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=potato) failed to identify a potato expressed sequence tag (EST) with sequence similarity to published CCD8 sequences. In order to identify the potato CCD8 gene homologue, a tBLASTx search (Altschul et al., 1990) of an early release of the potato genome was carried out using the Arabidopsis transcript sequence (AT4G32810.1) as a reference. Sequences showing a high degree of similarity were identified corresponding to amino acid sequences towards both the N- and C-terminal sequences (on scaffold St.Ac.001.Scaffold001141, 2009-06-05 BGI). The nucleotide sequences corresponding to the well-conserved amino acid sequences were used for primer design (see the 'Materials and Methods' section) and a 1368-nucleotide cDNA fragment was amplified from the root of potato cv Desiree. The deduced amino acid sequence of the amplified fragment was 68.9% identical to the Arabidopsis CCD8 sequence. Subsequently, the cDNA sequence was compared with the potato genome sequence (PGSC_DM_V3_2.1.10 pseudomolecule updated 15/12/2011 and available online at http://potatogenomics.plantbiology.msu.edu) and was shown to correspond to the single-copy CCD8 gene on chromosome 8 (Gene number PGSC0003DMG400002234). Examination of the RNAseq data for the doubled monoploid DM1-3 516R44 (DM) potato (publically available on the potato genome browser http://potatogenomics.plantbiology.msu.edu) indicated that the CCD8-specific transcript was expressed at very low levels in the DM potato type compared with the diploid line RH89-039-16 (RH) of cultivated potato. Subsequent comparison of the Desiree cDNA sequence with the tomato sequence available from the Sol Genomics Network website (http://solgenomics.net/) revealed a very high degree of sequence identity (96.3%) with the tomato CCD8 sequence (transcript number Solyc08 g066650.2.1; Kohlen et al., 2012). In order to obtain the full-length cDNA sequence from Desiree, 5′ and 3′ sequences were amplified from root cDNA using primers based on the tomato transcript sequence (Table S1).
The complete potato CCD8 (StCCD8) gene was predicted to have six exons and 3802 nucleotides (Fig. 1a). The coding sequence of the potato CCD8 gene is 1674 bp. As part of the characterization of the potato CCD8 gene, a sequence alignment was carried out, using the tomato SlCCD8 and petunia PhCCD8 protein sequences (Fig. S2). Next, a phylogenetic tree was generated using the CCD8 protein sequences from a variety of plant species (Fig. 1b). This analysis confirmed the taxonomic predictions, as StCCD8 clustered together with SlCCD8 and PhCCD8 in a well-supported subclade.
The expression of the potato CCD8 gene
A qRT-PCR assay was designed to measure the StCCD8 transcript level in a range of potato cv Desiree tissues (Fig. 2a; primer sequences were outside the RNAi hairpin sequence, as in the 'Materials and Methods' section). The maximum level of expression was observed in root tissues, up to four-fold lower levels in stem and barely detectable levels in flowers and leaves. In stolons, a relatively high level of expression was observed, which increased in swelling stolons at the onset of tuberization. However, in developing tubers, StCCD8 expression was up to 10-fold lower than in swelling stolons.
Down-regulation of CCD8 expression in CCD8-RNAi lines
In order to investigate the role of the StCCD8 gene in potato, transgenic plants were generated in which CCD8 expression was down-regulated. Hence, a 530-bp sequence of the StCCD8 cDNA (Fig. S1) was introduced into the pHellsgate8 vector under the control of a CaMV 35S promoter (Helliwell et al., 2002). The resulting StCCD8-RNAi construct was transformed into potato cv Desiree by Agrobacterium tumefaciens-mediated transformation, resulting in 20 independent transgenic potato lines. Two transgenic lines presenting a severe phenotype, when compared with wild-type potato, were selected for further analysis (lines 1 and 8). These lines exhibited similar phenotypes, and line 1 was selected and used consistently throughout the experiments described in this article. The StCCD8 expression of lines 17 and 21 was decreased less severely (Fig. 2), and therefore these were included in some of the experiments. The effect of transformation with the StCCD8-RNAi construct on gene expression was determined by qRT-PCR in tubers, roots and stems of potato. As transgene expression was driven by a constitutive 35S CaMV promoter, the accumulation of the StCCD8-specific transcript in all tissues examined was up to 10-fold less than in the wild-type controls, as was expected (Fig. 2b–d).
Increased shoot branching from main stem and stolons in StCCD8-RNAi lines
Phenotypic differences between the wild-type and StCCD8-RNAi potato were visible early in the development of lines RNAi-1, RNAi-8, RNAi-17 and RNAi-21, as nodes of the transgenic potato gave rise to shoots (Fig. 3a), which continued to grow into numerous lateral stems. Below ground, only a few stolons were observed as, instead of normal diageotropic growth, stolons tended to emerge and form aerial shoots. The nodes from the lower stems of transgenic potato plants were seen to develop miniature tubers (Fig. S3). These ‘aerial tubers’ produced shoots, not only from the apical bud, but also from lateral ones, indicating a perturbation of apical dominance (Fig. 3b). In addition, a reduction in plant height was observed for all RNAi plants (Fig. 3b). Transgenic potato lines showed a significant increase in the number of main and lateral stems (Fig. 3c) and reduced plant height (Fig. 3d). Although control plants produced flowers, no floral organs were formed in StCCD8-RNAi lines 1 and 8.
Increased secondary tuber formation and reduced dormancy in StCCD8-RNAi lines
Underground, the RNAi plants from lines 1 and 8 produced few stolons, with new tubers forming from sessile buds derived from mother tubers (Fig. 4a,b). Moreover, the fully underground tubers manifested an outgrowth of lateral buds, leading to an elongated and knobby tuber, not seen in control plants (Fig. 4c,d). The resulting tubers were elongated, similar in shape to microtubers of DM (Fig. S4). In pot experiments, the tuber yield of the StCCD8-RNAi plants was up to three-fold lower than in controls, with a large number of small-sized tubers (up to 2.9-fold more) formed by the transgenics (Fig. S5a,b). Tubers from the StCCD8-RNAi lines showed a higher degree of sprouting during storage than controls, with 100% of CCD8-RNAi tubers forming sprouts up to 0.3 cm in length after 12 wk of storage at room temperature, whereas no sprouting was observed in controls (Fig. S6).
Reduced chlorophyll accumulation in StCCD8-RNAi lines
The leaves of transgenic StCCD8-RNAi lines were of a paler green colour, suggesting that these plants had less chlorophyll than controls (Fig. S7a). This was confirmed by analysis of total chlorophyll, which showed lower levels in the leaves of CCD8-RNAi plants compared with controls (Fig. S7b).
Carotenoid levels in StCCD8-RNAi lines
To establish whether the StCCD8 gene can influence carotenoid accumulation in potato tubers, the total carotenoid content was determined spectrophotometrically in root and developing/mature tuber tissue. In developing tubers, total carotenoid levels were increased significantly in StCCD8-RNAi lines 1 and 8, the lines that expressed CCD8 at the lowest level, as confirmed by data from two growing seasons (Figs 5, S8a). However, in line 17, the carotenoid levels differed in developing tubers from 3 μg g−1 DW (first season) to 6.5 μg g−1 DW (second season of growth). Although differences were observed in developing tubers, the analysis of mature tubers failed to show any significant difference between the total carotenoid levels of RNAi transgenic potato plants and controls (Fig. S8b). In root tissue, where StCCD8 was found to be maximally expressed, no significant differences in the carotenoid content were found (Fig. S8c). Analysis by HPLC of the carotenoid composition of saponified and nonsaponified tissue extracts, in samples from two selected RNAi lines, did not show any difference between the major carotenoids accumulating in the StCCD8-RNAi lines and control samples (data not shown).
Reduced SL levels in StCCD8-RNAi lines
In most plants, the synthesis of SLs is thought to take place in the root and, possibly, the lower part of the stem (reviewed by Dun et al., 2009a). This is consistent with the expression data of StCCD8 in potato cv Desiree. The highest expression of the StCCD8 gene was in roots, but it was also expressed to a significant level in stolons. SLs are normally present in plants in minute amounts, and hence are very difficult to quantify (Lopez-Raez et al., 2008; Roumeliotis et al., 2012). In order to measure the SL levels in transgenic potato roots, the plants were grown under phosphate starvation conditions, known to facilitate the production and/or exudation of this phytohormone (Yoneyama et al., 2007; Lopez-Raez et al., 2008). Previously, orobanchol and orobanchyl acetate have been detected in potato roots (Roumeliotis et al., 2012). In the present study, orobanchol was the only SL present at levels that could be quantified accurately. The removal of phosphate from the nutrient solution increased the levels of orobanchol in control potato plants. This was not the case for the StCCD8-RNAi plants, lines 1 and 17, which were found to have up to 15-fold lower levels of orobanchol throughout the experiment (Fig. 6a).
GR24 complements the StCCD8-RNAi in vitro tuberization phenotype
A complementation experiment was devised to confirm that the observed phenotype is caused by reduced SL levels. For this, tubers were produced in vitro from wild-type and RNAi nodal cuttings of potato stem (Fig. 6b). In the absence of added SL, the RNAi tubers exhibited a distinct stem branching phenotype. Instead of a microtuber forming in the bud axil, either from a short stem or as a sessile tuber as in controls, there was a prolific growth of highly branched stems growing from the axillary bud. Tuberization was observed at both the terminal stem bud and axillary buds. The inclusion of the synthetic SL analogue GR24 in the solid medium had little effect on the tuberization of wild-type and empty vector samples. However, for the RNAi lines, the application of 1–10 mg l−1 GR24 in the medium resulted in a decrease in the extent of stem growth and degree of branching. At 10 mg l−1, the RNAi lines produced microtubers morphologically similar to those of the control samples, indicating that the phenotype of the StCCD8-RNAi transgenic potato lines was caused by decreased levels of SL.
Low level of StCCD8 expression in DM
Considerable RNAseq data are available for both RH and DM potato types that were the focus of the potato genome sequencing effort (http://potatosequence.org/). Whereas RH expresses the StCCD8-specific transcript in many tissues at high levels, expression in DM is barely detectable, making it likely that SL biosynthesis is compromised in this genotype. Comparison of the DM sequence with the Desiree cDNA sequence did not reveal any premature stop codons in the DM sequence. Using a series of PCR primers based on the DM and tomato CCD8 sequences, the genomic sequence from Desiree was amplified, cloned and sequenced. A number of insertions and deletions, both within the promoter region and within introns, were revealed by this analysis (Fig. S9). These differences in gene structure may account for the low StCCD8 expression level in DM. However, as the SL biosynthesis and response pathway is not a simple linear pathway and involves complex feedback regulatory mechanisms (Dun et al., 2009b) the reduced StCCD8-specific transcript level in DM may reflect differences elsewhere in the regulatory network.
Some of the phenotypic characteristics of DM appear similar to the StCCD8 mutant phenotype (Fig. S4 compared with Fig. 4d; particularly the elongated shape and outgrowth of tubers from axillary buds) and, together with the severely reduced StCCD8 expression, raised the question of whether the DM phenotype could be altered in the tuberization system as with the StCCD8-RNAi lines. Similar to the StCCD8-RNAi lines, in the absence of added SL, stem growth from explants was rapid and stems were highly branched. Inclusion of 10 mg l−1 in the medium resulted in much shorter stems with reduced branching (Fig. 6c), a result similar to that observed with the StCCD8-RNAi lines (Fig. 6b).
In vitro sprouting response in StCCD8-RNAi lines
SLs play an important role in the reduction of bud outgrowth, either directly (Brewer et al., 2009) or in combination with auxin (Crawford et al., 2010; Liang et al., 2010). The effect of StCCD8 down-regulation on tuber bud dormancy was investigated through a convenient system described as the sprout release assay (Hartmann et al., 2011). Using this procedure, the formation of sprouts was observed in buds excised from StCCD8-RNAi tubers. Tubers were harvested from 12-wk-old plants and then stored in darkness at 4°C for 2 wk. Monitoring the total percentage of sprout formation in water did not demonstrate any significant difference between RNAi lines and controls (Fig. S10).
CKs and gibberellins (GAs) are considered as sprout growth-promoting hormones (Suttle, 2004; Beveridge & Kyozuka, 2010). To establish how SLs affect the action of these growth regulators in potato buds, tuber discs from the sprout release assay were treated with GA3 or the synthetic CK, BAP. In wild-type potato, a high percentage of buds formed sprouts after 7 d of treatment with GA3 or BAP (100% and 42%, respectively; Fig. 7a,c), confirming previous reports (Hartmann et al., 2011). This effect was reduced significantly by the addition of GR24 to the treatment (Fig. 7b,d). Surprisingly, the RNAi transgenic lines were much less responsive than controls to GA3, as treatment induced up to 40% sprouting, compared with 100% in the wild-type (Fig. 7a). However, the RNAi lines responded in a similar manner to the wild-type on treatment with BAP (Fig. 7c). This suggests a decreased sensitivity to GA3, but not CK, in the StCCD8-RNAi transgenic potato lines.
BAP- or GA3-induced stolon growth associated with a rapid decline in StCCD8 mRNA
To gain more information on the expression pattern of the StCCD8 gene during sprouting, qRT-PCR analysis was performed, using sprouting and nonsprouting tuber buds of potato cv Desiree. As shown in Fig. 7(e), the StCCD8 transcript was detected in nonsprouting buds, immediately following water treatment, but was absent when samples from 3 and 6 d were analysed. However, when a sprout-inducing treatment was applied, containing 50 μM of GA3 and BAP, the StCCD8 gene transcript was not detected at the zero hour time point taken immediately after the 5-min pretreatment, suggesting a rapid down-regulation of StCCD8 expression by sprout-inducing treatments.
Expression of StTCP-1 is decreased in StCCD8-RNAi roots
Recently, it has been demonstrated that TEOSINTE BRANCHED1-CYCLOIDEA and PCF (TCP) transcription factors act downstream of SLs to control shoot branching in pea (Braun et al., 2012). In potato, a TCP gene, termed StTCP-1, is expressed at high levels in endodormant tuber apical buds, but, on dormancy release, expression of the StTCP gene is not detectable (Faivre-Rampant et al., 2004). The levels of StTCP-1 transcript were compared in roots from Desiree and the StCCD8-RNAi-1 line. In roots from both developing and mature plants, there was a significant decrease in StTCP-1 expression in StCCD8-RNAi roots compared with controls, similar to the decrease in transcript level of the pea TCP transcription factor PbBRC1 in the pea rms1 mutant (Braun et al., 2012; Fig. 8).
Recent research has demonstrated that SLs are involved in a far wider range of biological functions than just the regulation of above-ground shoot architecture (reviewed in Brewer et al., 2013). For example, a small-molecule screen in Arabidopsis identified several putative functions for SLs, including roles in seed germination and hypocotyl elongation (Tsuchiya & McCourt, 2010). In this study in potato, the phenotypes exhibited by the RNAi lines were complex and provide new insights into the roles of SLs, particularly in aspects of the potato tuber life cycle.
Potato SL levels relate to StCCD8 expression levels
Analysis of SL biosynthesis genes shows that CCD8 may be a key regulator of SL levels, as this gene is highly responsive to induction by environmental cues, such as low inorganic phosphate (Pi) (Kohlen et al., 2012). Some of the characteristics observed in the potato StCCD8-RNAi lines were similar to those seen in other species in which CCD8 expression has been reduced using RNAi approaches. For example, in tomato, CCD8-RNAi lines produced floral organs of reduced size (Kohlen et al., 2012). In potato, this effect appeared to be more severe, and StCCD8-RNAi lines did not produce any flowers. In StCCD8-RNAi lines, there was a marked decrease in chlorophyll content compared with controls, consistent with the observation of mild leaf chlorosis in dad1-1 mutants (Napoli, 1996). Complementation experiments and direct SL assays provide strong evidence that SL levels relate to StCCD8 expression levels in potato. Interestingly, the doubled monoploid potato DM that has been sequenced does not express StCCD8. In an in vitro tuberization system, DM shares aspects of its phenotype with the StCCD8-RNAi lines, which can be rescued by inclusion of synthetic SL in the tuberization medium. Therefore, interpretation of the DM RNAseq data should take into account the observation that DM might be compromised in its ability to produce SLs. As mutations in all SL biosynthetic genes give rise to highly branched, dwarf phenotypes, characteristic of plants impaired in SL biosynthesis (Brewer et al., 2013), similar phenotypes in potato may be elicited by the down-regulation of the potato orthologues of DWARF27, CCD7 and MAX1. Similarly, reduced expression levels of genes involved in SL perception, such as the orthologues of MAX2 and DAD2, may also elicit similar phenotypes.
SLs involved in diageotropic growth
A distinct feature of the StCCD8-RNAi lines was the growth habit of stolons. The diageotropic nature of stolon growth was lost in the StCCD8-RNAi lines, so that, instead of growing horizontally from the main stem, the majority of stolons emerged to produce above-ground stems. This implies that SL production in the stolon is necessary for the maintenance of diageotropic growth. Previously, it has been demonstrated that SLs are able to modulate local auxin levels, and that the net result of SL action is dependent on the auxin status of the plant (Brewer et al., 2009; Hayward et al., 2009; Ruyter-Spira et al., 2011). It is possible that the tightly balanced auxin–SL interaction is the basis for the mechanism of diageotropic stolon growth, and disruption of the level of SL can therefore have a major effect on this aspect of growth.
Reduced dormancy in tubers from StCCD8-RNAi lines
Possibly as a result of the lack of underground stolons, a high level of aerial tuber formation was observed in the StCCD8-RNAi lines. The buds from the aerial tubers exhibited a low degree of dormancy and produced sprouts as the aerial tubers were developing, not only from the apical bud, but also from axillary buds, indicating a total loss of apical dominance in the aerial tubers. It has been demonstrated recently that potato tubers exhibit apical dominance behaviour that is very similar to that of other stems (Roumeliotis et al., 2012; Teper-Bamnolker et al., 2012). Shoot apical dominance and branching regulation are thought to involve three long-range hormonal signals – auxin, SLs and CKs, although the details of their interaction remain to be fully clarified. Recent advances have described how CKs and SLs interact to impact on bud outgrowth (Dun et al., 2012). Evidently, the perturbation of StCCD8 expression and, by implication, the SL level has a dramatic effect on tuber apical dominance. Recently, it has been demonstrated that TCP transcription factors act downstream of SLs to control shoot branching (Braun et al., 2012; Dun et al., 2012). Expression of the StCCD8 gene was high in dormant buds, but its expression level decreased on treatment with hormones known to cause release from dormancy. This pattern of expression is consistent with a role for SLs in the regulation of TCP expression, which, in turn, may control the activation status of the meristem. In root samples, there was a small, but significant, decrease in StTCP-1 expression in StCCD8-RNAi lines, similar to the decrease in transcript level of the pea TCP transcription factor PbBRC1 in the pea rms1 mutant (Braun et al., 2012; Fig. 8). StCCD8-RNAi tubers exhibited a lower degree of tuber dormancy compared with parental controls and empty vector lines – typically sprout growth to > 0.3 cm was observed in 100% of the RNAi tubers, whereas 100% of controls remained fully dormant. Perhaps the observed lack of expression of StCCD8 in the DM potato may indicate that alleles of CCD8 that are not expressed are relatively common in potato germplasm. Conceivably, these alleles have been retained during domestication as they might confer low tuber dormancy, important when rapid tuber growth cycles are required. However, the low levels of StCCD8 expression in DM may be a result of other factors that regulate CCD8 expression (Mashiguchi et al., 2009), and a genetic investigation of the co-segregation of the phenotype and specific CCD8 alleles will be required to substantiate this hypothesis.
SLs affect GA3-induced sprout release
When compared using the sprout release assay, significant differences in tuber sprouting behaviour were found between buds from the transgenic lines and controls. In wild-type Desiree, sprouting was stimulated by GA3 treatment, so that close to 100% sprouting was observed after 7 d – a result similar to that reported previously by Hartmann et al. (2011). However, in GA3-treated buds from RNAi lines, only c. 40% of buds had sprouted after 7 d. There are very few studies documenting an involvement of GAs in the control of apical dominance. Early research showed a role for GAs in the promotion of auxin-derived inhibition of bud growth in decapitated plants of pea (Jacobs & Case, 1965; Scott et al., 1967). There are also two reports on a negative correlation between bioactive GA1 levels and branching, as seen in Citrus and Chrysanthemum transgenic plants (Fagoaga et al., 2007; Miao et al., 2010). Interestingly, it has been proposed recently that GAs could change bud sensitivity to application of synthetic SLs (Luisi et al., 2011), as seen in dwarf pea plants with low GA1. Our results also suggest an interaction between SL levels and bud sensitivity to GA3. GR24 treatment inhibited tuber sprout growth, and this inhibition could not be overcome by either GA3 or BAP treatment, providing evidence of SLs acting downstream of these other phytohormones.
The sprout release assay was shown to be a suitable system to study sprouting behaviour (Hartmann et al., 2011), but it is also important to consider the effects of wounding-associated ethylene, produced when the buds are excised from tubers, on sprouting. Interaction between SLs, auxin and ethylene has been demonstrated in different systems (Kapulnik et al., 2011; Rasmussen et al., 2012). Currently, little is known about this interaction in potato, with the study of Hartmann et al. (2011) suggesting that the ethylene-associated signals negatively influence the growth of sprouts.
SLs affect carotenoid levels
Measurements of the total carotenoid content in developing tubers of the StCCD8-RNAi lines with the lowest StCCD8 expression level showed an up to 2.5-fold increase when compared with controls. However, in mature tubers and roots of all StCCD8-RNAi lines tested, there were no changes in carotenoid levels compared with controls. This suggests an increased rate of carotenoid accumulation in StCCD8-RNAi developing tubers compared with controls, but apparently both reach the same end level of accumulated carotenoids. Our understanding of the role of CCDs in dictating carotenoid accumulation has increased greatly recently. Microarray experiments in potato have clearly shown enhanced expression of CCD4 in white-fleshed potato tubers, with much lower expression levels of CCD4 in carotenoid-accumulating yellow-fleshed potato tubers (Ducreux et al., 2008; Campbell et al., 2010). Silencing CCD7 and/or CCD8 expression in tomato (Vogel et al., 2010), kiwi fruit (Ledger et al., 2010) and Chrysanthemum flowers (Liang et al., 2010) did not result in any change in total carotenoid levels, as observed in mature tubers of StCCD8-RNAi potato plants. It is not known whether the rate of carotenoid accumulation is different in the different developing structures of tomato, kiwi and chrysanthemum, as observed for developing tubers of StCCD8-RNAi potato plants (Figs 5, S8a).
Although it is believed that, as SLs are produced in low quantities, a loss of SL biosynthesis might not affect carotenoid levels significantly, it might be that these differences are more tissue specific than previously thought. In developing tubers, consistent increases in total carotenoid content were observed in strongly down-regulated StCCD8-RNAi lines. This could be explained by a consideration of the pathway for SL biosynthesis (Alder et al., 2012). Thus, all-trans-β-carotene is isomerized by D27 to yield 9-cis-β-carotene, a product which is then cleaved by CCD7 at the C9′, C10′ position into 9-cis-β-apo-10′-carotenal. Then, CCD8 converts this product to carlactone, an SL-like compound. Considering that all-trans-β-carotene is a substrate for CCD7 cleavage, effects on downstream carotenoid levels are possible, and suggest that the carotenoid pool size is determined, at least in part, by the activity of CCDs.
Overall, we conclude that SLs play an important role in dictating the architecture of potato plants and also in maintaining tuber bud dormancy. The StCCD8-RNAi plants, deficient in SL biosynthesis, produced tubers that sprouted earlier than controls. In addition, the RNAi tuber buds showed a diminished response to application of GA3 sprout-inducing treatment, indicating a relationship between SL and GAs that requires further investigation.
This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Division, EU-FP7 METAPRO 244348 and a short-term scientific mission to S.A.P. from COST Action FA1006, The Plant Engine.