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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.
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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.