Knockdown of strigolactone biosynthesis genes in Populus affects BRANCHED1 expression and shoot architecture

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1 Research Knockdown of strigolactone biosynthesis genes in Populus affects BRANCHED1 expression and shoot architecture Merlin Muhr, Nele Pr ufer, Maria Paulat and Thomas Teichmann Department of Plant Cell Biology, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, G ottingen 3777, Germany Author for correspondence: Thomas Teichmann Tel: tteichm@gwdg.de Received: 2 April 216 Accepted: 25 May 216 New Phytologist (216) 212: doi: /nph.1476 Key words: axillary meristem, branching, bud outgrowth, MAX4, poplar, Populus 9 canescens, strigolactones, tree architecture. Summary Plant architecture is modified by a regulatory system that controls axillary bud outgrowth. Key components in this system are strigolactones (SLs) and BRANCHED1, which inhibit bud outgrowth. Their role has been described in herbaceous model systems, including Arabidopsis, rice and pea. However, a role in woody perennial species, including the model tree poplar, has not been unequivocally proven. In this study, we tested a role for SLs in Populus 9 canescens by treatment with the synthetic SL GR24. We generated MORE AXILLARY BRANCHING4 (MAX4) knockdown lines to study the architectural phenotype of poplar SL biosynthesis mutants and the expression of SLregulated genes. We show that GR24 is perceived by the model tree poplar. MAX4 knockdown lines exhibit typical SL deficiency symptoms. The observed changes in branching pattern, internode length and plant height can be rescued by grafting. We identified putative poplar BRANCHED1 and BRANCHED2 genes and provide evidence for a regulation of BRANCHED1 by SLs. Our results suggest a conservation of major regulatory mechanisms in bud outgrowth control in the model tree poplar. This may facilitate further research, pinpointing the role of SLs and BRANCHED1 in the complex regulation of bud outgrowth in trees. Introduction Plants exhibit a high degree of phenotypic plasticity and their architecture is variable. The embryos and seedlings contain the shoot and root apical meristem as primary meristems, which establish a simple unbranched shoot and root as the primary growth axis. During further shoot development, axillary (secondary) meristems grow in leaf axils. They can form axillary buds, which become dormant or grow out to a branch as a secondary growth axis (Bennett & Leyser, 26). Thus, axillary buds can profoundly change the plant appearance and architecture, but their activity is strictly regulated. The plant apex suppresses bud outgrowth via apical dominance, a mechanism for whose establishment the plant growth hormone auxin is necessary and sufficient (Thimann & Skoog, 1934; Phillips, 1975; Cline, 1997; Leyser, 25). However, as auxin does not need to enter the bud, apical auxin suppresses bud outgrowth indirectly (Hall & Hillman, 1975). Two theories were established to explain this indirect action. According to the auxin canalization theory, an auxin flux that emanates from an auxin source towards an auxin sink allows the establishment of a polar auxin transport stream (PATS) (Sachs, 1981; Bennett et al., 214). This appears to be crucial for a subsequent connection of the bud with the vascular system as a prerequisite for bud outgrowth. Thus, with respect to auxin, the gradient between the source strength of the bud and the sink strength of the stem determines bud outgrowth potential Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust (reviewed in Domagalska & Leyser, 211). The second messenger theory is an alternative model to explain the indirect effect of auxin on bud outgrowth. It claims that auxin controls synthesis or transport of a component that transmits the auxin signal from the apex into the axillary bud (Snow, 1937). Cytokinin was identified as a potential second messenger. Biosynthesis of this bud outgrowth promoting hormone is negatively regulated by auxin (Sachs & Thimann, 1967; Nordstrom et al., 24; Tanaka et al., 26). However, recent analyses show that cytokinin signaling cannot explain all aspects of bud outgrowth control (Mueller et al., 215). Additional likely second messengers are the recently discovered strigolactones (SLs), which suppress bud outgrowth (Gomez- Roldan et al., 28; Umehara et al., 28). Auxin enhances the expression of SL biosynthesis genes (Hayward et al., 29) and SLs may subsequently relay the primary auxin signal into the bud. However, the inhibitory effect of SLs on bud outgrowth may also be explained by the auxin transport canalization model. SLs were found to decrease the amount of the auxin efflux carrier PIN at the plasma membrane and, consequently, reduce PATS (Shinohara et al., 213). Such a reduction would enhance dormancy in axillary buds by lowering the auxin sink strength of the stem. In addition to these two models implying that SLs are acting by controlling auxin fluxes or as second messengers of auxin, analyses of Brewer et al. (215) show that in pea, changes in auxin transport or auxin concentrations are not necessary for bud outgrowth. The authors suggest a direct action model, which New Phytologist (216) 212:

2 614 Research New Phytologist implies that SLs directly control downstream targets, such as expression of the transcription factor BRC1, independent of upstream auxin signaling. Regardless of the exact mode of action, the highly branched phenotype of SL biosynthesis or signaling mutants reflects their prominent role in the control of bud outgrowth. SLs are a diverse group of structurally related molecules synthesized from carotenoids. The first steps of SL biosynthesis take place in plastids. An all-trans/9-cis-b-carotene isomerase (DWARF27 (D27)) in rice, Arabidopsis and Medicago (Lin et al., 29; Waters et al., 212a; van Zeijl et al., 215) catalyzes the isomerization of alltrans-b-carotene. The resulting 9-cis-b-carotene is converted to carlactone in two steps by the carotenoid cleavage monoxygenases CCD7 and CCD8, which have been designated MORE AXILLARY BRANCHING3 (MAX3) and MORE AXILLARY BRANCHING4 (MAX4) in Arabidopsis (Sorefan et al., 23; Booker et al., 24). The corresponding orthologs in pea are RAMOSUS5 (RMS5) and RAMOSUS1 (RMS1) (Sorefan et al., 23; Johnson et al., 26), DWARF17 (D17) and DWARF1 (D1) in rice (Ishikawa et al., 25; Arite et al., 27; Alder et al., 212) and DECREASED APICAL DOMINANCE3 (DAD3) and DECREASED APICAL DOMINANCE1 (DAD1) in petunia (Snowden et al., 25; Drummond et al., 29). Carlactone may be a mobile intermediate of SL biosynthesis that is transported acropetally (Seto & Yamaguchi, 214). In Arabidopsis, carlactone is converted to carlactonic acid by MAX1, a cytochrome P45 monooxygenase (Abe et al., 214). In rice, five MAX1 paralogs exist and one of these catalyzes the conversion of carlactone to the SL orobanchol (Zhang et al., 214). The SL signaling pathway is not fully understood either. It is now generally accepted that an a/b-fold hydrolase (D14 in Arabidopsis and rice, DAD2 in petunia; Arite et al., 29; Hamiaux et al., 212; Waters et al., 212b) is important for SL perception. Via an F-box protein (MAX2 in Arabidopsis, RMS4 in pea, MAX2A in petunia and D3 in rice; Stirnberg et al., 22; Ishikawa et al., 25; Johnson et al., 26; Drummond et al., 212), SL signaling leads to the degradation of downstream target proteins. Although D53 was identified as a target in rice (Jiang et al., 213; Zhou et al., 213), details of the downstream pathway are still unknown. It is well established that SLs can act directly within the bud by positively regulating transcript abundance of the bud outgrowth repressor BRANCHED1 (BRC1) (Aguilar-Martinez et al., 27; Braun et al., 212; Dun et al., 212). BRC1 belongs to the class II of TB1 CYCLOIDEA PCF (TCP) type transcription factors (Aguilar-Martinez et al., 27; Finlayson, 27), which act as cell cycle inhibitors (Martin-Trillo & Cubas, 21). Owing to a genome duplication (Franzke et al., 211; Vanneste et al., 214), Arabidopsis contains the BRC1 paralog BRC2. However, mainly BRC1 controls bud outgrowth in Arabidopsis, as indicated by the findings that brc1 loss-of-function mutants are highly branched, while brc2 knockout plants exhibit a weaker phenotype (Aguilar- Martinez et al., 27; Finlayson, 27). The significance of the SL pathway for branching control has been demonstrated for the annual plants Arabidopsis, pea and rice and the perennials petunia and kiwifriut (Stirnberg et al., 22, New Phytologist (216) 212: ; Sorefan et al., 23; Umehara et al., 28; Ledger et al., 21; Dun et al., 213). There is also evidence supporting a role for SLs in branching control in trees. Ward et al. (213) carried out complementation experiments of Arabidopsis max knockout mutants using the respective orthologs from Salix viminalis hybrids and Salix aurita. They showed that willow Sx-MAX1, Sx- MAX2 and Sx-MAX4 fully complement the branching phenotype of the respective Arabidopis mutants, with the exception of Sx- MAX4 from S. aurita, which only partially reverted the high branching phenotype. This indicates that MAX4 allelic polymorphisms exist in the genus Salix which may correlate with differences in branching patterns and resprouting after harvest (coppicing). Indeed, Salmon et al. (214) observed that the coppicing response of willow depends on the presence of a specific willow MAX4 allele. Similar to the studies in willow, Czarnecki et al. (214) showed that expression of poplar MAX orthologs reduces the branching phenotype of Arabidopis max mutants. Studies by Zheng et al. (216) suggest that in addition to genes involved in SL biosynthesis, components of SL signaling are also present in poplar. Complementation analyses in the Arabidopsis d14 mutant using two poplar genes with high homology to the SL receptor DWARF14 (D14) identified one functional D14 ortholog in poplar. Moreover, Agusti et al. (211) observed a stimulation of secondary growth in eucalyptus trees treated with the synthetic SL analog GR24. Taken together, these data suggest that the SL pathway also exists in trees and indicate that SL biosynthesis may affect tree performance in short rotation coppices. However, direct proof using transgenic trees with altered SL biosynthesis or signaling has not been obtained yet. Also the role of BRC1 as a SL-regulated gene has not been addressed in trees. Using amirna constructs, we generated transgenic poplar lines with a significant down-regulation of both poplar MAX4 orthologs. Phenotypic analyses, grafting experiments and auxin transport assays suggest SL deficiency of the obtained lines and demonstrate the significance of SL for branching control in trees. In addition, we analyzed putative poplar BRC1 and BRC2 orthologs and provide data pointing to a functional diversification of these transcriptional repressors in poplar. Materials and Methods Plant material and growth conditions Populus 9 canescens (Aiton) Sm. INRA 717-1B4 was used as the wild-type. Wild-type and transgenic plantlets were propagated in vitro on half-strength MS medium (Duchefa Biochemie BV, Haarlem, the Netherlands) supplemented with 2% (w/v) sucrose by preparing stem cuttings. The plantlets were cultivated in a growth chamber under a 16 : 8 h, light: dark, photoperiod at 22 : 18 C, 6% relative humidity. Lighting at 7 8 lmol m 2 s 1 PAR was provided by fluorescent tubes (type 84 and Fluora, Osram GmbH, München, Germany). If required, plantlets were transferred to soil (Fruhstorfer Erde Typ T25, Hawita Gruppe GmbH, Vechta, Germany) supplemented with 5% (v/v) washed screed sand (/8 mm) and cultivated further in a growth chamber or a greenhouse. In greenhouse Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust

3 New Phytologist Research 615 experiments, a minimum temperature of 22 : 14 C, day : night, was maintained by heating. Natural daylight was supplemented by metal halide lamps (HQI-TS 25W/D; Osram) to maintain a 16 h photoperiod. To provide outdoor conditions, plants were grown in a greenhouse covered with a wire mesh (no heating and artificial lighting). Transformation of P. 9 canescens Populus 9 canescens was transformed using Agrobacterium tumefaciens according to a transformation protocol adopted from Matthias Fladung, Th unen Institute of Forest Genetics, Großhansdorf, Germany. Shoots of c. 6-wk-old P. 9 canescens wild-type plantlets grown in vitro under standard conditions were cut into small explants and incubated in Agrobacterium culture (grown to OD 6 =.5) for 3 min at 28 C. Explants were cocultivated with Agrobacterium for 3 d in the dark on Petri dishes containing half-strength MS medium supplemented with 2% sucrose and subsequently distributed on Petri dishes containing medium supplemented with kanamycin (5 mg l 1 ), cefotaxime (15 mg l 1 ), ticarcillin clavulanate (2 mg l 1 ) and thidiazuron (.22 mg l 1 ). Regenerates developing after 2 6 wk were transferred into culture vessels for further growth. When shoots emanated from the regenerates, cuttings were made and placed on selective medium without thidiazuron for rooting. The transgenic status of rooted cuttings was verified by PCR. To produce high numbers of genetically identical individuals of each independent primary transgenic plant, stem cuttings were prepared and propagated in vitro. Phenotypic analyses of architectural parameters Adventitious rooting was assessed by growing wild-type and transgenic plantlets in vitro under standard conditions. The total number of formed adventitious roots was counted c. 8 wk after preparation of the cuttings. For phenotyping of shoot architectural traits, plants were grown on soil in a growth chamber, a greenhouse or a greenhouse covered with a wire mesh to provide outdoor conditions, as indicated in the corresponding experiment. The number of branches, the number of nodes and the shoot height were determined. GR24 treatment of P. 9 canescens stem cuttings Stem cuttings bearing two nodes (including the bud, the leaf was removed) were prepared from in vitro grown P. 9 canescens plantlets. Cuttings were randomized and placed on standard medium containing the synthetic SL analog rac-gr24 (Chiralix BV, Nijmegen, the Netherlands) or the same volume of solvent (acetone;.5% final concentration). Bud outgrowth was defined as the unfolding of the first leaf and monitored daily. Auxin transport assay Twelve millimeter segments of internode 6 (counted from the apex) were excised from the main stems of soil-grown Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust Populus 9 canescens wild-type and MORE AXILLARY BRANCHING4 knockdown plants (representative lines T14#4A and T22#5A) using a razor blade. The segments were placed in plastic tubes containing 1 ll of a.5% MES buffer solution supplemented with 18 lm 14 C-radiolabeled IAA (1 lci ml 1 14 C-IAA; American Radiolabeled Chemicals Inc., St Louis, MO, USA). The apical ends were facing downwards, that is, the solution was applied apically. Negative controls were performed with inverted internodes (basal end in solution) as well as addition of 3 lm a-naphthylphthalamic acid (Naptalam Pestanal, Sigma- Aldrich). Samples were incubated for 5 h at 22 C. The basal end (2 mm; apical end for inverted control) of each internode was excised using a razor blade, weighed and used for determination of radioactivity levels as a quantification of transported 14 C-IAA. Poplar grafting In vitro grown plantlets were transferred to soil and acclimatized. The stem of the rootstock was cut horizontally at 4 5 cm above the soil level using a scalpel blade. The stump was sliced vertically in the middle, c. 5 mm deep. The scion was prepared by cutting the apex of an appropriate plant at a length of 2 3 cm. All leaves > 1 cm length were removed to reduce transpiration and the stump of the scion was cut to form a wedge shape. The scion was inserted into the sliced stump of the rootstock. The graft union was mechanically stabilized with silicone tube (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and wrapped in Parafilm M laboratory film (Bemis Inc., Neenah, WI, USA) to prevent drying. The grafted plants were cultivated under high humidity. For the first day after grafting, the plants were kept entirely dark, and shading was successively removed over the course of 1 wk. Then, the plants were acclimatized to lower humidity. After 2 3 wk, the plants formed a stable graft union and were further cultivated in a greenhouse for phenotyping of architectural parameters. Phylogenetic analyses Putative poplar BRANCHED1 and BRANCHED2 orthologs were identified in a TBLASTN search using the Arabidopsis thaliana BRANCHED1 (BRC1) and BRANCHED2 (BRC2) full protein sequences as query against the Populus trichocarpa genome on Phytozome ( A CLUSTALW alignment of the full protein sequences of all Arabidopsis TCP proteins (including BRC1 and BRC2) (Martin- Trillo & Cubas, 21) from The Arabidopsis Information Resource ( and all poplar TBLASTN hits was performed using the software MEGA5.2 (Tamura et al., 211). A phylogenetic tree was constructed in MEGA5.2 based on the neighbor-joining method (1 bootstrap replicates). Evolutionary distances were calculated using the Poisson correction method. The CYC-TB1 clade (Martin-Trillo & Cubas, 21) was identified and all Arabidopsis and poplar TCP protein sequences from this clade were used to build a phylogenetic tree as described earlier. As additional references, the protein sequences of BRC1 orthologs in pea (Pisum sativum Ps-BRC1; GenBank: AEL1223.1), tomato (Solanum lycopersicum Sl- New Phytologist (216) 212:

4 616 Research New Phytologist BRC1A (Solyc3g11977) and Sl-BRC1B (Solyc6g6924)) and rice (Oryza sativa Os-TB1; Os3g4988), as well as snapdragon CYCLOIDEA (Antirrhinum majus Am-CYC; GenBank: CAA ), were included. amirna design and cloning For the design of artificial microrna (amirna) constructs to specifically knock down the selected candidate genes, the WMD3 Web MicroRNA Designer tool ( cgi-bin/webapp.cgi) (Schwab et al., 26) was used. The P. trichocarpa reference sequences of the target genes were used as input, and the corresponding amirna sites were designed using default settings. The highest-ranked amirnas (best fit to targets and low probability of off-targets, based on P. trichocarpa genome sequence information) were considered. Since P. 9 canescens was used in this work, the target sites of the amirnas were confirmed in the corresponding sequences obtained from the Aspen Database ( The mirna and mirna sites of the endogenous poplar microrna precursor ptr-mir48 (Lu et al., 25) were replaced with the designed sequences to obtain a candidate gene-specific amirna. For the MAX4a MAX4b double knockdown, the amirna 5 -TAAGGAATTATGAACCTGCCG-3 and the amirna 5 -CGACAGGTTCATATTTCCTTT-3 were successfully used. The amirna construct was designed with flanking Gateway attl sites and ordered as synthetic DNA construct in the puc57 vector from Genscript USA Inc. (Piscataway, NJ, USA). The construct was transferred into the binary Gateway destination vector pk7wg2 (Karimi et al., 22) by performing a LR reaction. RNA extraction and expression analysis Samples were taken from in vitro- or soil-grown plants as indicated in the corresponding experiments. From in vitro-grown plantlets, whole stems without leaves were used. From soil-grown plants, bark, developing xylem and wood were sampled at the main stem. The bark was peeled off manually. Developing xylem was scraped off the stem with a scalpel blade. The remaining tissue was taken as a wood sample. For expression analyses in leaves, the first fully expanded leaves were taken from the apex. Root samples were taken from young, vital roots after washing off the soil. Axillary buds were harvested from nodes 3 to 3 (counted from the apex). All samples were snap-frozen in liquid nitrogen and stored at 8 C until processing. A CTAB-based protocol (Chang et al., 1993) was used for RNA extraction from different poplar tissues. RNA yield from dormant axillary buds can be low. Therefore, an improved extraction protocol was applied for RNA preparation from dormant axillary buds from wild-type and plants used for poplar BRC1 and BRC2 candidate gene expression analysis. Instead of following the standard procedure (Chang et al., 1993), the RNA was further purified with a kit (innuprep Plant RNA Kit, Analytik Jena) after LiCl precipitation, as suggested by Ruttink et al. (27). Within one experiment, all samples were extracted using the same protocol. Genomic DNA was removed New Phytologist (216) 212: by DNase I treatment (Thermo Fisher Scientific Inc., Waltham, MA, USA). cdna synthesis from 1 lg of total RNA was performed with the RevertAid H Minus First Strand cdna Synthesis-Kit (Thermo Fisher Scientific Inc.) using an oligo-dt primer. Primers used for quantitative PCR (qpcr) are listed in Supporting Information Table S1. Poplar UBIQUITIN (Pc-UBQ) was used as reference gene (Regier & Frey, 21). The reaction setup (15 ll total volume) was done using the SsoFast EvaGreen Supermix (Bio-Rad). Primers were provided at a final concentration of 5 nm each. As template,.6 ll of the cdna were added. For each reaction, three technical replicates were made. The reaction was performed according to the manufacturer s recommendations on a CFX96 Real-Time PCR Detection System (Bio-Rad). The quantification cycle (Cq) was determined for all samples. The cycle number difference between the target and reference genes [DCq(target-reference)] was calculated and the relative expression was determined according to the formula 2 Dcq. The number of replicates indicated in the figure legends refers to the number of biological replicates (plants). Each sample was processed individually. Statistical analysis Statistics were evaluated with GRAPHPAD ( QuickCalcs t-test Accession numbers Sequence information of genes from P. trichocarpa described in this article is filed in the Plant Comparative Genomics portal of the Department of Energy Joint Genome Institute ( under the following accession numbers: PtMAX4a, Potri.18G441; PtMAX4b, Potri.6G2385; putative BRANCHED orthologs, Potri.12G599; Potri.1G132; Potri.8G1158 and Potri.15G55; UBQ, Potri.1G4185. Results Strigolactone suppresses bud outgrowth in poplar To test whether SLs are functional suppressors of bud outgrowth in the model tree poplar, stem cuttings comprising two axillary buds were treated with the synthetic SL GR24. This two-node assay was chosen because results from Arabidopsis, chrysanthemum (Dendranthema grandiflorum) and willow (Salix sp.) suggest that competition between two buds, each representing an auxin source, is necessary to observe the inhibitory effect of SLs without external application of apical auxin (Crawford et al., 21; Liang et al., 21; Ward et al., 213). While in Arabidopsis outgrowth of both the upper and basal buds was inhibited by GR24 (Crawford et al., 21), a specific inhibition of only the basal bud was observed in chrysanthemum and willow (Liang et al., 21; Ward et al., 213). Also in poplar, there was no significant effect at the apical bud in GR24-treated cuttings, but we observed a significant reduction of Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust

5 New Phytologist Research 617 outgrowth of the basal bud (Fig. 1), showing that GR24 can suppress bud outgrowth in poplar. Generation of MAX4 knockdown () lines For further analysis of the relevance of SLs in poplar, we designed artificial microrna (amirna) constructs to down-regulate expression of the key SL biosynthesis gene MAX4 in Populus 9 canescens. The two MAX4 orthologs PtrMAX4a (Potri.18G441) and PtrMAX4b (Potri.6G2385) of P. trichocarpa have been previously identified and analyzed through complementation of Arabidopsis max4 mutant phenotypes (Czarnecki et al., 214). Transcripts of both target genes were detected in wood samples (stem without bark and developing xylem) when expression was monitored in various samples of greenhouse-grown P. 9 canescens wild-type plants. Interestingly, expression of MAX4a and MAX4b in other tissues was low or not detectable (Fig. 2a). As experiments by Czarnecki et al. (214) indicate that both individual poplar orthologs are functional, redundancy among both was expected. For this reason, we performed simultaneous knockdowns of MAX4a and MAX4b and generated 14 independent transgenic lines (). Transcript abundances of MAX4a and MAX4b in representative lines were significantly reduced compared with the wild-type, confirming successful knockdowns (Fig. 2b). Noticeably, residual transcript abundances were significantly higher in line T22#13A relative to the other knockdown lines. Thus, although there still was a significant reduction of transcripts relative to the wild-type, the knockdown was less efficient. MAX4 knockdown lines show increased branching and other typical SL-deficiency symptoms For phenotypic analysis, all 14 generated double knockdown lines were grown on soil in a greenhouse. While the P. 9 canescens wild-type plants did not produce any branches (a) (b) 1 under these conditions when grown to a height of c. 1.5 m, all transgenic lines developed lateral branches and most lines exhibited reduced shoot height. Five representative lines were chosen for a quantitative characterization of shoot phenotypes. The poplar lines exhibited significantly increased branch numbers compared with wild-type plants (Fig. 3a). In addition, the plant height and average internode length were significantly reduced in most of the tested lines (Fig. 3b,c). The habitus of typical wild-type and plants is shown in Fig. 3(e). Line T22#13A developed fewer branches than the other knockdown lines and showed wild-type height and internode length. Thus, the extent of phenotypic alterations correlates with target gene knockdown efficiency (Figs 2b, 3b,c). These changes in morphology of poplar MAX4 knockdown lines match the phenotypes of SL biosynthesis mutant plants of other species (Beveridge et al., 1996; Napoli, 1996; Stirnberg et al., 22; Sorefan et al., 23; Ishikawa et al., 25; Snowden et al., 25; Arite et al., 27; de Saint Germain et al., 213) and indicate SL deficiency. To test the stability of the phenotypes at different growth conditions, the representative lines T14#4A and T22#5A were grown under controlled conditions in a climate chamber, as well as in a greenhouse covered with a wire mesh to provide outdoor conditions. As the phenotypes were reproducible (Fig. S2), we used the two best-characterized lines T14#4A and T22#5A for further experiments. In addition to their role in shaping shoot architecture, SLs were reported to influence root growth. For instance, adventitious root formation was reported to be increased in Arabidopsis, pea and rice SL pathway mutants (Rasmussen et al., 212; Sun et al., 215). Thus, we analyzed adventitious root formation in stem cuttings of representative poplar MAX4 knockdown lines compared with the wild-type. The MAX4 knockdown lines showed significantly increased adventitious rooting (Fig. 3d), suggesting that this function of SLs is conserved in trees as well. Another effect of SLs is the inhibition of auxin transport in Arabidopsis and, consequently, Arabidopsis SL deficiency mutants exhibit increased auxin ns µm GR24 5 µm GR24 Bud outgrowth rate (%) Medium (+GR24) Fig. 1 GR24 treatment inhibits bud outgrowth in Populus 9 canescens stem cuttings. In vitro grown wild-type stem cuttings bearing two nodes were prepared and placed on growth medium containing lm (mock) or 5 lm of the synthetic strigolactone GR24 (a). The outgrowth rate 1 d after preparation of the cuttings was determined individually for the upper and lower buds (b). Data represent means SD (n = 3 culture vessels containing seven to eight cuttings each). Significant differences with Student s t-test between mock- and GR24-treated samples are indicated:, P <.1; ns, not significant. The experiment was repeated twice and representative data are shown. Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust New Phytologist (216) 212:

6 618 Research New Phytologist (a).14 (b) nd nd nd nd nd nd MAX4a MAX4b MAX4a MAX4b P. x can T14 #1A T14 #4A T14 #6A T22 #5A T22 #13A Fig. 2 Populus 9 canescens (P 9 can) MAX4 orthologs are expressed in the stem (a) and are down-regulated in artificial microrna (amirna) knockdown lines (b). Expression of the Populus 9 canescens MORE AXILLARY BRANCHING4 orthologs MAX4a and MAX4b was determined in different tissues of wild-type () plants grown on soil relative to the reference gene UBIQUITIN (UBQ) (a). The highest expression was found in wood samples, while transcripts were not detectable ( nd ) in leaf, axillary bud and bark samples. Data represent means SD (n = 6 plants). For confirmation of an efficient simultaneous knockdown of MAX4a and MAX4b, expression was quantified in wood samples of soil-grown Populus 9 canescens plants and five independent MAX4 knockdown lines () relative to UBQ (b). Significant differences with Student s t-test between and the knockdown lines are indicated (, P <.1). Although significantly reduced relative to the, transcript abundances in line T22#13A were significantly higher relative to the other knockdown lines, indicated by. Lines T14#4A and T22#5A were chosen as representative lines for additional experiments in this work and successful knockdown of MAX4a and MAX4b was confirmed twice in independent experiments. Expression strength of MAX4 genes strongly depends on age or position of the internode (Supporting Information Fig. S1). As a consequence, total expression values vary between experiments depending on the age of the plants at harvest (compare Fig. S1a and b). Expression data from one experiment were obtained from tissues of the same age and position. Dev. xylem, developing xylem. transport rates (Shinohara et al., 213). This phenotype was addressed in the poplar MAX4 knockdown lines, too. Indeed, representative lines show a small, but significant increase of polar 14 C IAA transport from the apical to the basal ends of excised stem segments (Fig. 4). New Phytologist (216) 212: Shoot phenotypes of MAX4 knockdown lines can be complemented by grafting to wild-type rootstocks Summarized, our experiments indicate that SLs play a role in controlling various aspects of poplar architecture. We successfully generated poplar MAX4 knockdown lines, which exhibit typical root and shoot architectural phenotypes of SL-deficient plants. However, direct confirmation of SL deficiency was not feasible, because SLs are difficult to quantify as a result of their low concentration and high instability (Akiyama & Hayashi, 26). Furthermore, SLs are structurally diverse (Yoneyama et al., 29; Zwanenburg et al., 29; Xie et al., 21) and yet unknown SLs may exist in trees. For these reasons, we performed grafting experiments to obtain further indirect evidence for SL deficiency in plants. Similar experiments were already used in different species before the discovery of SLs as branchingsuppressing hormones, showing that the substance suppressing bud outgrowth can be synthesized in roots and be transported acropetally from a wild-type rootstock into a SL-deficient scion. There, it can suppress the increased branching phenotype of SLdeficient mutants (Napoli, 1996; Foo et al., 21; Sorefan et al., 23). Also in our analysis in poplar, wild-type rootstocks were able to fully complement increased branching in scions (Fig. 5). The other observed shoot phenotypes of plants, that is, the reduced plant height and internode length, were also fully complemented (Fig. S3). Thus, the wildtype rootstock seems to be able to synthesize SLs, which appear to be transported acropetally as observed in other species. A wild-type scion grafted to an rootstock also exhibited a wild-type-like phenotype (Figs 5, S3), indicating that the poplar shoot appears to be sufficient as a site for SL biosynthesis and does not depend on a product of MAX4 supplied by the root. Identification of poplar BRC1 and BRC2 candidate genes Arabidopsis BRC1 and, to a lower extent, BRC2, as well as orthologs in other species, are transcription factors that function as central regulators of bud outgrowth. They integrate various signals influencing bud outgrowth, including SLs (Doebley et al., 1997; Takeda et al., 23; Kebrom et al., 26; Aguilar-Martinez et al., 27; Finlayson, 27; Martin-Trillo et al., 211; Braun et al., 212; Drummond et al., 215). Hence, characterization of poplar BRC orthologs may contribute to the analysis of the signaling chain controlling bud outgrowth in trees. Here, we report on the identification of BRC1 and BRC2 candidate genes in the model tree poplar. Candidate genes were identified by protein sequence comparison with Arabidopsis BRC1 and BRC2 and are shown in a phylogenetic tree (Fig. 6). The poplar BRC1 and BRC2 candidate genes were further characterized by expression analysis. A hallmark of BRC1 and BRC2 is their bud-specific expression in Arabidopsis. While transcripts are abundant in dormant axillary buds, expression in other tissues was found to be low or absent. Only in floral organs was considerable expression reported (Aguilar-Martinez et al., 27; Finlayson, 27). Here, we tested expression of the poplar Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust

7 New Phytologist Research 619 (a) Number of branches nd (b) Plant height (cm) (c) Average internode length (cm) (d) Number of adventitious roots (e) Fig. 3 Populus 9 canescens (P. 9 can) lines exhibit typical phenotypes of strigolactone-deficient mutants. Relative to the wild-type (), knockdown lines of both poplar MORE AXILLARY BRANCHING4 orthologs () exhibit significantly increased branch numbers (a), a significantly increased shoot height (b) and internode length (c), as well as a significantly increased number of adventitious roots (d). (e) The habitus of representative wild-type () and (line T14#4A) specimens. (a c, e) Phenotyping data of soil-grown plants from an individual experiment (n = 3 8 plants). (d) Phenotyping data from in vitro grown plants (n = 15). Data represent means SD. Significant differences with Student s t-test between the and the knockdown lines are indicated:, P <.5;, P <.1;, P <.1. nd, not detectable. All experiments were repeated twice and representative data are shown. candidate genes in different tissues of greenhouse-grown P. 9 canescens wild-type plants. The expected tissue specificity was observed for one of the two BRC1 and BRC2 candidate genes each. Potri.12G599 and Potri.1G132 expression was high in dormant axillary buds, but low or undetectable in other tissues (Fig. 7). Expression of the other candidate genes was generally low and less specific. Another characteristic of Arabidopsis BRC1 is a reduction of transcript abundance in outgrowing buds, fitting to its role as a suppressor of bud outgrowth (Finlayson, 27). To test this characteristic expression pattern in poplar, dormant buds from greenhouse-grown wild-type plants were sampled along with a series of growing buds at different stages of bud elongation, induced by decapitation. The expected reduction of expression in elongating buds was only observed for the two BRC1 and BRC2 Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust candidate genes Potri.12G599 and Potri.1G132, respectively (Fig. 8a e). In summary, two poplar BRC1 and BRC2 candidate genes each were identified by phylogenetic analysis. One candidate gene each, Potri.12G599 and Potri.1G132, exhibits characteristic bud-specific expression and down-regulation in elongating buds. Expression of the poplar BRC1 candidate genes is reduced in MAX4 knockdown lines BRC1 was reported to be regulated by SLs in herbaceous species. Its transcript abundances were found to be reduced in Arabidopsis, pea and petunia SL pathway mutants. Furthermore, GR24 treatment increased BRC1 expression in pea (Aguilar- New Phytologist (216) 212:

8 62 Research New Phytologist (a) Basal Internode 6 Apical 14 C IAA 18 μm 2 mm 1 mm (b) 14 C radioac vity [kbq g 1 FW] Inverted 3 μm NPA P. x can T14 #4A T22 #5A Controls () Fig. 4 Populus 9 canescens (P. 9 can) lines have an enhanced auxin transport capacity in the stem; 12 mm segments of internode 6 (counted from the apex) were excised from the stems of soil-grown wild-type () and MORE AXILLARY BRANCHING4 knockdown (, representative lines T14#4A and T22#5A) plants (a). The apical ends were placed in a 14 C IAA solution. Negative controls were performed with inverted internodes (basal end in solution) and the auxin transport inhibitor a-naphthylphthalamic acid (NPA). After 5 h, 14 C radioactivity was determined in the basal end (2 mm; apical end for inverted control) of each internode (b). Data represent means SD (n = 6 plants). Significant differences with Student s t-test between the wild-type and the knockdown lines are indicated:, P <.5;, P <.1. The experiment was repeated twice and representative data are shown. Number of branches by the use of the MAX4 knockdown lines generated in this study, which exhibit typical SL deficiency symptoms. Expression was monitored in dormant axillary buds of representative lines. Compared with the wild-type, expression of both BRC1 candidate genes (Potri.12G599 and Potri.15G55) was significantly reduced in MAX4 knockdown lines (Fig. 8f,g), suggesting a positive regulation of these genes by SLs in wild-type poplar. By contrast, expression of the BRC2 candidate genes (Potri.8G1158 and Potri.1G132) was not altered (Fig. 8h,i). 2 nd Scion T14 #4A T14 #4A T22 #5A T22 #5A Rootstock T14 #4A T14 #4A T22 #5A T22 #5A Fig. 5 The shoot branching phenotype of Populus 9 canescens MAX4 knockdown lines can be complemented by grafting. Rootstock scion graft combinations of the wild-type () and MORE AXILLARY BRANCHING4 knockdown lines (, representative lines T14#4A and T22#5A) were prepared. Branch numbers were determined for soil-grown plants. The upper label indicates the genotype of the scion, while the lower label specifies the genotype of the rootstock. Data represent means SD (n = 8 plants). Significant differences with Student s t-test between the wild-type and the knockdown lines are indicated:, P <.1. nd, not detectable. The experiment was repeated twice and representative data are shown. Martinez et al., 27; Braun et al., 212; Drummond et al., 215). By contrast, BRC2 expression does not appear to be SLresponsive, as it was not changed in Arabidopsis SL pathway mutants. Also in petunia, not all BRC-like orthologs appeared to be SL-regulated (Aguilar-Martinez et al., 27; Drummond et al., 215). To further characterize the poplar BRC1 and BRC2 candidate genes, we investigated their regulation by SLs. This was facilitated New Phytologist (216) 212: Discussion In this study, we show that bud outgrowth in the model tree poplar can be suppressed by the synthetic SL GR24. SL biosynthesis knockdown () lines exhibit a variety of SL deficiency-associated phenotypes and the shoot architectural phenotypes can be rescued by grafting. Furthermore, we identified BRANCHED1 and BRANCHED2 candidate genes in poplar and provide evidence that expression of the BRANCHED1 candidate genes is controlled by SLs. SLs suppress bud outgrowth in poplar To corroborate the hypothesis that SLs control shoot architecture in poplar, we cultivated poplar wild-type stem cuttings bearing two axillary buds on medium containing GR24. The treatment significantly inhibited outgrowth of the lower bud in poplar (Fig. 1). Inhibition of outgrowth of the lower bud and unaffected outgrowth of the upper bud were also observed in chrysanthemum and willow (Liang et al., 21; Ward et al., 213). Possibly, the bud that is apical in a two-node assay has been closer to establishing its own PATS in the intact plant. Thus, in a two-node assay, the upper bud may be more competitive and GR24 Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust

9 New Phytologist Research At-BRC1 (At-TCP18) Ps-BRC1(24.9 %) Os-TB1 (24.4 %) Potri.12G599 (24.5 %) Potri.15G55 (25.3 %) Sl-BRC1B (23. %) Sl-BRC1A (25.8 %) At-BRC2 (At-TCP12) Potri.1G132 (26.1 %) Potri.8G1158 (28. %) At-TCP1 Potri.17G112 (24.9 %) Am-CYC (28.1 %) Fig. 6 Phylogenetic tree of putative Populus trichocarpa BRANCHED1 and BRANCHED2 orthologs. Poplar candidate genes were identified in a TBLASTN search using the Arabidopsis thaliana BRANCHED1 (BRC1) and BRANCHED2 (BRC2) full protein sequences as query against the Populus trichocarpa genome on Phytozome ( A CLUSTALW alignment of the full protein sequences of all Arabidopsis TCP proteins (including BRC1 and BRC2) and all poplar TBLASTN hits was performed using the software MEGA5.2 (Tamura et al., 211). A phylogenetic tree was constructed in MEGA5.2 based on the neighborjoining method. The tree clearly reflected the Class I and Class II clades of TCP transcription factors, with Class II being separated into the subclades CIN and CYC/TB1 (Martin-Trillo & Cubas, 21). All Arabidopsis and poplar TCP protein sequences from the CYC-TB1 clade were used to build the tree shown here (neighbor-joining method, MEGA5.2). As additional references, the protein sequences of BRC1 orthologs in pea (Pisum sativum Ps-BRC1), tomato (Solanum lycopersicum Sl-BRC1A and Sl- BRC1B) and rice (Oryza sativa Os-TB1), as well as snapdragon CYCLOIDEA (Antirrhinum majus Am-CYC), were included. Bootstrap test values (1 replicates) are indicated. The sequence identity relative to the corresponding Arabidopsis protein (BRC1, BRC2 and At-TCP1) is indicated in brackets. exclusively inhibits the lower bud. The data show that GR24 is perceived by the model tree poplar and suggest a conserved role of SLs in bud outgrowth control in poplar. Analyses of MAX4 knockdown lines provide evidence that SLs control bud outgrowth in poplar To further investigate the function of SLs in trees, we knocked down both poplar MAX4 orthologs (described in Czarnecki et al., 214) to disrupt SL biosynthesis. Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust To choose the correct tissue for analysis of knockdown efficiency, gene expression was monitored in soil-grown wild-type plants. We found clearly detectable MAX4 expression in the wood of the stem, albeit at a low level, and only minor amounts of MAX4 transcripts in the root and developing xylem (Fig. 2a). In contrast to these findings, strong At-MAX4 promoter activity was found consistently in Arabidopsis roots, while only weak activity was observed in the hypocotyl, petioles and nodal sections of the inflorescence (Sorefan et al., 23; Bainbridge et al., 25). Moreover, comparable amounts of MAX4 transcripts were detected in both the stem and roots of rice (Oryza sativa), chrysanthemum (D. grandiflorum) and petunia (Petunia hybrida) (Sorefan et al., 23; Snowden et al., 25; Arite et al., 27; Liang et al., 21). Thus, the very low level of MAX4 expression in poplar roots as an expected site of SL biosynthesis appears to be surprising. However, grafting experiments in Arabidopsis, pea and petunia revealed that the root as a site for SL biosynthesis is not required at least for SL functions in the shoot, as wild-type scions grafted on max4 mutant rootstocks develop a wild-type branching pattern (Napoli, 1996; Foo et al., 21; Sorefan et al., 23). The results of our grafting experiments with poplar MAX4 knockdown rootstocks and wild-type scions support the notion that local SL biosynthesis in the stem is sufficient for bud outgrowth inhibition (Figs 5, S3). The comparably high expression of MAX4 genes in the poplar stem may even be a necessity for the sufficient supply of SLs to the stem of trees. Czarnecki et al. (214) reasoned that as trees are much taller than herbaceous plants, transport of SLs from the root to the shoot along large distances may not be sufficient to control shoot branching. While local MAX4 activity in SL biosynthesis in the shoot therefore appears to have a more prominent role in poplar compared with other (nontree) species and root-derived MAX4 products do not seem to play a major role in the shoot, MAX4 expression is not entirely absent from poplar roots (Fig. 2a). Therefore, MAX4 activity in roots may still be important in the control of local traits, such as root architecture and signaling in the rhizosphere. When expression of the target genes was monitored in stem samples of knockdown lines, a significant down-regulation of both MAX4 genes was confirmed (Fig. 2b). Four of the five investigated lines exhibit a comparably strong down-regulation of the MAX4 genes and show typical SL-deficiency phenotypes (Fig. 3). Line T22#13A developed fewer branches and showed no changes in internode length and plant height. The fact that residual MAX4 transcript abundances are significantly higher in line T22#13A compared with the other amimax4 lines shows that the observed architectural changes correlate with MAX4 expression levels, and thus, most likely, SL content. In pea and petunia, SLs were reported to stimulate internode elongation by increasing the cell division rate (Snowden et al., 25; de Saint Germain et al., 213). As a consequence, SLdeficient mutants are smaller. The control of internode length by SLs seems to be conserved in poplar as most of the analyzed MAX4 knockdown lines exhibit a reduced internode length compared with wild-type plants (Fig. 3c). In addition to these shoot phenotypes discussed, SLs are also known to be involved in the control of root growth. Rasmussen New Phytologist (216) 212:

10 622 Research New Phytologist Potri.12G599 Potri.15G55 Potri.8G1158 nd Potri.1G132 Fig. 7 Expression of Populus 9 canescens BRANCHED1 and BRANCHED2 candidate genes is bud-specific. Expression of the candidate genes was determined in different tissues of soil-grown wild-type plants. Data represent means SD (n = 4 plants). nd, not detectable. et al. (212) reported increased adventitious rooting of Arabidopsis and pea SL pathway mutants. This was confirmed in poplar MAX4 knockdown lines (Fig. 3d). Moreover, lines exhibit increased auxin transport rates (Fig. 4), indicating that the effects of SLs on auxin transport in poplar are comparable to the effects in Arabidopsis (Shinohara et al., 213). Thus, taking all findings together, these lines revealed shoot architecture and root formation phenotypes as well as physiological alterations that are typical for SL-deficiency mutants. This indicates that the function of SLs in controlling many aspects of plant growth is conserved in the model tree poplar. Poplar BRC1 candidate genes are putative targets of SLs In a phylogenetic analysis, Potri.15G55 and Potri.12G599 were identified as poplar BRC1 candidate genes, while Potri.8G1158 and Potri.1G132 are probably BRC2 orthologs (Fig. 6). Thus, there are two candidates each for BRC1 and BRC2, probably resulting from a genome duplication event that occurred in the Salicaceae family (Tuskan et al., 26). Expression analysis was performed to identify likely functional poplar BRC orthologs based on typical expression patterns. This includes high expression in dormant axillary buds and downregulation upon bud outgrowth in Arabidopsis (Aguilar-Martinez et al., 27; Finlayson, 27). Potri.12G599 is highly expressed in axillary buds, but was also detected in other tissues (Fig. 7). This is similar to the expression pattern of BRC1 in Arabidopsis, in which northern blot and qpcr data revealed low New Phytologist (216) 212: transcript abundance in various tissues (Aguilar-Martinez et al., 27; Finlayson, 27). By contrast, Potri.1G132 shows a lower expression rate, but is almost exclusively expressed in axillary buds (Fig. 7). Interestingly, Potri.12G599 and Potri.1G132 not only show bud-specific expression, but also exhibit down-regulation during axillary bud outgrowth (Fig. 8). The maximal decreases in expression of Potri.12G599 and Potri.1G132 during bud outgrowth are 45% and 65%, respectively. These are comparable to expression changes in other plants under conditions that favor bud outgrowth. After decapitation of Arabidopsis plants, AtBRC1 shows a reduction in transcript abundance of 6% and AtBRC2 shows a reduction of 5% (Aguilar-Martinez et al., 27). Also, the tomato orthologs of AtBRC1 exhibit a 45 65% decrease after decapitation (Martin-Trillo et al., 211). The difference in TB1 expression between modern maize cultivars and highly branched teosinte is 5% (Doebley et al., 1997). By contrast, changes in light conditions or light perception that suppress bud outgrowth increase AtBRC1 expression by 3 6% and AtBRC2 expression by 5% (Finlayson et al., 21; Gonzalez-Grandio et al., 213). The observed expression characteristics clearly discriminate Potri.12G599 and Potri.1G132 from their paralogs. Potri.12G599 and Potri.1G132, respectively, exhibit expression characteristics typical for BRC1 and BRC2. In Arabidopsis, pea and petunia, the expression strength of BRC1 corresponds to SL concentrations (Aguilar-Martinez et al., 27; Braun et al., 212; Drummond et al., 215). Therefore, we expected reduced expression of BRC1 candidate genes in axillary buds of the SL-deficient lines. Indeed, transcript Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust

11 New Phytologist Research 623 (a) Potri.12G599 (f) Potri.12G Status: Dormant 2 3 mm 3 5 mm 5 12 mm > 12 mm P. x can T14 #4A T22 #5A (b).16 Potri.15G55 (g).5 Potri.15G Status: Dormant 2 3 mm 3 5 mm 5 12 mm > 12 mm P. x can T14 #4A T22 #5A (c).45 Potri.8G1158 (h) Potri.8G Status: Dormant 2 3 mm 3 5 mm 5 12 mm > 12 mm.2.1 P. x can T14 #4A T22 #5A (d).6 Potri.1G132 (i).4 Potri.1G Status: Dormant 2 3 mm 3 5 mm 5 12 mm > 12 mm P. x can T14 #4A T22 #5A (e) Dormant 2 3 mm 3 5 mm 5 12 mm > 12 mm Fig. 8 Expression of Populus 9 canescens (P. 9 can) BRANCHED1 and BRANCHED2 candidate genes is reduced in elongating buds and in dormant buds of MORE AXILLARY BRANCHING4 knockdown lines. Expression of poplar BRANCHED1 (Potri.12G599 and Potri.15G55) and BRANCHED2 (Potri.8G1158 and Potri.1G132) candidate genes was determined in dormant and elongating buds of soil-grown wild-type () plants relative to the reference gene UBIQUITIN (UBQ)(a d). Photographs of representative buds used for this experiment are shown in (e). Expression of the candidate genes was also monitored in dormant buds of soil-grown and MAX4 knockdown plants (; lines T14#4A and T22#5A) relative to UBQ (f i). The experiment was repeated twice and representative data are shown. Data represent means SD (n = 4 1 plants for (a d) and n = 5 plants for (f i)). Significant differences with Student s t-test between dormant and elongating buds (a d) or between and plants (f i) are indicated:, P <.5;, P <.1;, P <.1. Ó 216 The Authors New Phytologist Ó 216 New Phytologist Trust New Phytologist (216) 212: