Plant and Cell Physiology Advance Access originally published online on June 19, 2009
Plant and Cell Physiology 2009 50(8):1416-1424; doi:10.1093/pcp/pcp091
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d14, a Strigolactone-Insensitive Mutant of Rice, Shows an Accelerated Outgrowth of Tillers
1Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836 Japan
2RIKEN Plant Science Center, Tsurumi, Yokohama, 230-0045 Japan
3Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, 113-8657 Japan
4Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
*Corresponding author: E-mail, akyozuka{at}mail.ecc.u-tokyo.ac.jp; Fax, +81-3-5841-5087.
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Abstract |
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Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the
/β-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.
Keywords: DWARF 14 - Hormone signaling - Rice - Shoot branching - Strigolactone
Abbreviations: BAC, bacterial artificial chromosome; CCD, carotenoid cleavage dioxygenase; dCAPS, derived cleaved amplified polymorphic sequence; epi-5DS, 2'-epi-5-deoxystrigol; GUS, β-glucuronidase; HLS, hormone-sensitive lipase; LC/MS-MS, liquid chromatography-quadruple/time-of-flight tandem mass spectrometry; RT–PCR, reverse transcription–PCR; SA, salicylic acid; SABP2, salicylic acid-binding protein 2; SNP, single nucleotide polymorphism; SSR, simple sequence repeat; X-Gluc, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary dataFundingAcknowledgmentsReferences |
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The pattern of shoot branching is one of the critical determinants of the aerial plant architecture (Steeves and Sussex 1989
). Shoot branching starts from the generation of axillary buds in the axil of leaves. Axillary buds often become dormant after they are formed, and resume their outgrowth later in development. The fate of axillary buds, to grow or to remain dormant, is governed by the complex interplay of environmental and endogenous cues (McSteen and Leyser 2005).
In the last decade, remarkable progress has been made in understanding the molecular basis of the control of shoot branching through studies of a set of highly branched mutants isolated from pea, Arabidopsis and petunia (for reviews, see Beveridge 2006, Ongaro and Leyser 2008). Four mutants, max1–max4, that work in a single genetic pathway were identified in Arabidopsis and five mutants, ramosous1 (rms1) to rms5 were reported in pea. Extensive physiological analysis and grafting experiments of these mutants revealed the involvement of a graft-transmissible branch-inhibiting hormone in the control of shoot branching. Although the postulated hormone had not been identified, analysis of the mutants inferred that they were deficient in either the synthesis or signaling of the hormone. Among the four Arabidopsis genes, MAX1, MAX3 and MAX4 were predicted to be involved in the production of the hormone, while MAX2 was predicted to play a role in its perception or signaling. Similarly, in pea, RMS1 and RMS5 appear to be involved in the biosynthesis of the hormone while RMS3 and RMS4 would be involved in the signaling pathway of the hormone.
Molecular identification of MAX genes has demonstrated the validity of the prediction (Stirnberg et al. 2002, Sorefan et al. 2003, Booker et al. 2004, Booker et al. 2005). Consistent with the prediction that MAX1, MAX3 and MAX4 were involved in the synthesis of the hormone, they encode proteins that function as enzymes. MAX1 encodes a class III cytochrome P450, CYP711A1 (Booker et al. 2005). MAX3 and MAX4 encode carotenoid cleavage dioxygenases (CCDs), leading to the possibility that the branch-inhibiting hormone is synthesized from carotenoids (Sorefan et al. 2003, Booker et al. 2004). On the other hand, MAX2, a putative signaling component, encodes a member of the F box protein family that is often involved in the signal transduction of plant hormones (Stirnberg et al. 2002). RMS1, RMS4 and RMS5 were shown to be orthologous to MAX4, MAX2 and MAX3, respectively (Sorefan et al. 2003, Foo et al. 2005, Jhonson et al. 2006). Petunia Decreased apical dominance1 (DAD1) was also shown to be an ortholog of MAX4/RMS1 (Snowden et al. 2005), indicating that the regulatory pathway for the inhibition of branch shoot outgrowth is well conserved across different species.
Rice is another useful system to study the control of axillary bud outgrowth (Ishikawa et al. 2005). One advantage of using rice is that the phenotype of the branching mutants can be easily distinguished at early stages of plant development. Previously, we reported five tillering dwarf mutants, dwarf3 (d3), d10, d14, d17 and d27 (Ishikawa et al. 2005). D3 and D10 have been identified as orthologs of MAX2/RMS4 and MAX4/RMS1/DAD1, respectively, extending the conservation of the mechanism controlling shoot branching to monocots (Ishikawa et al. 2005, Arite et al. 2007). In addition, it was shown that high tillering dwarf1 (htd1) and d17 are mutant alleles of the rice MAX3/RMS5 ortholog (Zou et al. 2006, Umehara et al. 2008). Like RMS1 and DAD1, the D10 mRNA level is up-regulated in the mutants in which the branch-inhibiting hormone pathway is disrupted (Arite et al. 2007). D14 and D27 genes remain to be cloned and characterized; however, significant up-regulation of D10 expression was found in d14 and d27 as in d3, d10 and d17 mutants, implying that D14 and D27 most probably are involved in the same branch-inhibiting pathway.
The chemical nature of the predicted branch-inhibiting hormone was revealed recently from studies of pea and rice (Gomez-Roldan et al. 2008, Umehara et al. 2008). Application of strigolactones rescued mutant phenotypes of d10/rms1 and d17/rms5, hormone-deficient mutants, but not that of d3/rms4, putative signaling mutants. It was confirmed that endogenous strigolactones were indeed reduced to an undetectable level in roots of d10 and d17, whereas the level was elevated in d3, most probably as a result of a feedback regulation. Based on these results, it was concluded that strigolactones or their downstream metabolites constitute the mobile branch-inhibiting hormone. Strigolactones are a group of terpenoid lactones and are structurally related to strigol, the first member of this class of terpenes (Fig. 1). Prior to this discovery, strigolactones have been identified twice, first as a trigger of parasitic seed germination (Cook et al. 1972) and then as an inducer of mycorrhizal hypha branching (Akiyama et al. 2005). This discovery, combined with previous findings, suggested that strigolactones may act not only as inhibitors of shoot branching but also as mediators of nutrition uptake and the control of plant development.
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Regardless of the remarkable progress which has been made, still little is known about the molecular mechanisms by which strigolactones act to control shoot branching. Also, it has yet to be determined whether strigolactones are the active form of the hormone or if they are precursors that need to be converted to the bioactive form in order to function in the plant. Here, we report that rice d14-1 shows reduced sensitivity to strigolactones and accumulates elevated levels of endogenous strigolactones. Positional cloning revealed that D14 encodes a protein of the
/β-hydrolase superfamily. We propose that D14 is a novel player in the strigolactone pathway and functions at a late step of active hormone synthesis or in its signaling pathway. |
Results |
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d14 is a new strigolactone-insensitive mutant
d14 is a recessive tillering dwarf mutant (Ishikawa et al. 2005
). As we reported previously, D10 expression, which is subjected to a feedback regulation, is elevated in d14-1, suggesting that D14 probably functions in the strigolactone pathway along with D3, D10 and HTD1/D17 (Arite et al. 2007). To confirm this notion, we produced d10-1 and d14-1 double mutants and analyzed their phenotype. Both d10-1 and d14-1 show increased branching and dwarf phenotypes, and they are indistinguishable from each other in appearance (Ishikawa et al. 2005). The double mutant plants also exhibited the same phenotype as d10-1 and d14-1 (Fig. 2A, B), suggesting that D14 works in the same strigolactone pathway.
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To understand the role of D14, we examined whether the d14-1 mutant phenotype could be rescued by the application of GR24, a synthetic strigolactone analog (Fig. 2C). As reported previously, application of 1 µM GR24 to the hydroponic medium completely inhibited the outgrowth of the first and second axillary buds of d10-1, a strigolactone-deficient mutant (Umehara et al. 2008
). In contrast, growth of the first and second leaf buds of d14-1 was not suppressed by the same treatment with GR24. Application of GR24 to d10-1 d14-1 double mutant plants did not rescue the branching phenotype (Fig. 2C). The sensitivity of the d14-1 mutant to strigolactones was examined in more detail (Fig. 2D). The responses of d14-1 plants to (+)-strigol, a natural strigolactone, and GR24 were measured in parallel with other d mutants, and the results for d3-1, d10-1 and d17-1 mutants have been published previously (Umehara et al. 2008). Outgrowth of tillers in d14-1 was not suppressed by (+)-strigol or GR24, mimicking the responses of d3-1, a strigolactone-insensitive mutant (Umehara et al. 2008). This indicates that d14-1 has reduced strigolactone sensitivity. However, our current experiments do not distinguish whether d14-1 mutant plants are totally insensitive to strigolactones or whether their tiller bud outgrowth is still inhibited in response to higher doses of strigolactones.
Endogenous levels of 2'-epi-5-deoxystrigol (epi-5DS, Fig. 1), a native strigolactone of rice, were determined in roots by liquid chromatography-quadruple/time-of-flight tandem mass spectrometry (LC/MS-MS). We have previously reported that d3-1 roots accumulated much higher levels of epi-5DS than do wild-type roots when phosphate ion (P) is depleted in the medium, whereas epi-5DS is undetectable in d10-1 irrespective of the nutrient conditions (Umehara et al. 2008). We show here that the levels of epi-5DS in d14-1 roots and root exudates were significantly higher than those in wild-type roots under both P-rich and P-deficient conditions (Fig. 2E, F).
Next, we carried out a highly sensitive parasitic seed germination assay using Striga hermonthica seeds whose germination is induced by strigolactones (Fig. 2G). d14-1 root exudates in P-deficient medium showed a stronger germination-stimulating activity than did those of the wild type, in accordance with the increase in epi-5DS levels determined by LC-MS/MS.
Isolation of the D14 gene by positional cloning
We have isolated the D14 gene by map-based cloning. Rough mapping delimited the D14 locus within a 3 cM region on chromosome 3 (Fig. 3A). Subsequently, the locus was further narrowed to a 3.6 kb interval on bacterial artificial chromosome (BAC) clone AC146702
[GenBank]
by fine mapping. We compared sequences of the eight putative genes predicted in this region between the wild-type and d14-1. In the d14-1 mutant, a 615 bp insertion was found in an intron of a gene encoding a putative /β-hydrolase superfamily protein (Os03g0203200/AK070827) (Fig. 3B). The inserted sequence was shown to be an endogenous active transposon nDart1 (Tsugane et al. 2006) (Supplementary Fig. S1). Reverse transcription–PCR (RT–PCR) analysis showed that the mRNA of Os03g0203200 was below the level of detection in d14-1 (Fig. 3C), suggesting strongly that the insertion of nDart1 into Os03g0203200 is the cause of the d14-1 phenotype. To confirm this, we introduced a 4.8 kb genome fragment of Os03g0203200, containing the 2.7 kb upstream region, the open reading frame and the 1.0 kb downstream sequences, into d14-1. The transformed plants showed similar phenotypes to the wild type in both plant height and tiller development (Fig. 3D). The dominant co-segregation between the introduced genome fragment and complementation of the mutant phenotype was further confirmed in subsequent generations (data not shown). These results led to the conclusion that D14 corresponds to Os03g0203200.
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D14 is a member of the
/β-hydrolase superfamilyD14 encodes a protein of 318 amino acids that is classified as a member of the
/β-hydrolase superfamily (Supplementary Fig. S2). There is a closely related homolog of D14 (Os03g0437600) in the rice genome which we refer to as D14-like. Two D14 homologs (At3g03990 and At4g37470) were found in Arabidopsis, and phylogenetic analysis showed that they are orthologs of D14 and D14-like, respectively (Fig. 4). D14 homologs are found in a wide range of plant species including ferns (Selaginella moellendorfir) and bryophytes (Physcomitrella patens and Marchantia polymorpha). Interestingly, all D14 homologs in gymnosperm, fern and bryophyte species are classified into the D14-like subfamily, while the D14 subfamily consists of only genes from angiosperm species.
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The proteins of the
/β-hydrolase superfamily do not show significant sequence similarities but exhibit structural similarities, with three conserved amino acids, a nucleophilic residue, an acidic residue and a histidine residue in the catalytic center (Nardini and Dijkstra 1999, Ollis et al. 1992). Among the /β-hydrolase superfamily members, D14 and D14 homologs show a significant sequence similarity to Regulator of Sigma B (RsbQ) of Bacillus subtillis (Fig. 4). The three amino acids, called the catalytic triad, Ser147, Asp268 and His297, are also conserved in the D14 and D14 homologs (Supplementary Fig. S2).
Tissue specificity of D14 mRNA expression
Tissue specificity of D14 expression was examined by RT–PCR analysis. D14 mRNA was detected in all tissues examined except for root tips (Fig. 5A). Relatively high levels of D14 mRNA accumulation were detected in leaves and the first leaf buds. The spatial distribution of D14 gene expression was further examined by using the GUS (β-glucuronidase) gene as a reporter. An approximately 2.7 kb upstream region of D14, which was used in the complementation test, was used as a promoter to drive GUS. Three independent transgenic lines were used for analysis. GUS activity was low in all transgenic lines examined and variable in strength among different lines, but D14:GUS expression was consistent in that it is only detectable in the vasculature. Examination of transverse sections revealed that D14:GUS is expressed in parenchyma cells surrounding the xylem in leaves, stems and axillary buds (Fig. 5B–G).
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Discussion |
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The d14-1 mutant exhibits a dramatic increase in tiller numbers and a reduction in height, resembling the d3, htd1/d17 and d10 mutants (Ishikawa et al. 2005
, Zou et al. 2006). Here, we showed that the d10-1 d14-1 double mutants are indistinguishable from either single mutant, confirming that D14 indeed works in the strigolactone pathway. Furthermore, unlike the case with d10-1, phenotypic defects of the d14-1 mutant were not rescued by the application of (+)-strigol or GR24, indicating that d14-1 has reduced sensitivity to strigolactone. Consistent with this, d14-1 mutant plants accumulate an elevated level of epi-5DS, a major endogenous strigolactone, as often seen for hormone levels in hormone-insensitive mutants. Positional cloning of the D14 gene revealed that it encodes a protein of the /β-hydrolase superfamily. Based on these results, we propose that D14 is a new component in the strigolactone pathway and acts in a step downstream of the synthesis of strigolactones. D14 could function as the enzyme that catalyzes the metabolic conversion(s) of strigolactones to the as yet unknown bioactive form of the hormone or a component for the perception or transduction of the hormonal signal (Fig. 6). To determine whether D14 works in the biosynthesis or signaling of the hormone, grafting experiments could provide information. If a wild-type rootstock could rescue the phenotypic defects in a d14 scion this would suggest that D14 functions in the synthesis of the active hormone. However, unlike in Arabidopsis and pea, grafting of rice plants is technically very difficult. Therefore, analysis of Arabidopsis and pea D14 orthologs is important in order to examine whether D14 is a common component in the branch-inhibiting hormone pathway. Besides the grafting experiments, further biochemical studies of D14 will be necessary to elucidate its exact role in the strigolactone pathway.
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Members of the
/β-hydrolase superfamily exhibit similar overall 3D structures and possess the invariant catalytic triad: a nucleophilic residue, an acidic residue and histidine (Ollis et al. 1992, Nardini and Dijkstra 1999). Among the /β-hydrolase superfamily proteins, D14 resembles RsbQ of B. subtillis at the level of the amino acid sequence (Brody et al. 2001). RsbQ participates in the stress signaling cascade that leads to the activation of Sigma Factor B (SigB) through the activation of RsbP. Although the enzymatic activity of RsbQ is still under debate, crystallographic analysis of RsbQ demonstrated that it has a hydrophobic cavity that can potentially accommodate a small compound (Kaneko et al. 2005). The binding with this unknown small molecule is thought to be essential for its function. The catalytic triad and some residues that constitute the hydrophobic cavity in RsbQ (Phe27, Val97 and Phe196) are conserved in D14 and its homologs (Supplementary Fig. S2). The sequence and structural similarities between D14 and RsbQ suggest an intriguing idea that D14 might directly interact with strigolactones or downstream metabolites.
There is evidence that some members of the /β-hydrolase superfamily act as a high-affinity binding protein of small molecules (receptors) without performing a hydrolytic reaction. GID1, a soluble receptor of gibberellin, belongs to the /β-hydrolase superfamily with a similarity to hormone-sensitive lipases (HSLs) (Ueguchi-Tanaka et al. 2005). GID1 lacks the conserved histidine residue, which increases the nucleophilicity of the hydroxyl group of the serine (nucleophile) residue, in the catalytic triad of HSLs. Thus, GID1 does not possess hydrolase activity. Recent analysis of GID1 crystal structures revealed that, instead of acting as a nucleophile that attacks a carbonyl carbon to initiate hydrolysis, the conserved serine residue in the catalytic triad of GID1 is responsible for forming a hydrogen bond with the carboxyl group of gibberellins (Murase et al. 2008, Shimada et al. 2008). SABP2, a salicylic acid (SA)-binding protein, is another member of the /β-hydrolase superfamily (Kumar et al. 2003, Forouhar et al. 2005). Although SABP2 was initially purified as an SA-binding protein, it has the conserved catalytic triad and does function as an esterase that converts methyl salicylate to SA. SA is bound in the active site and acts as a potent inhibitor of the catalytic activity. It still has to be investigated further whether SA binding to SABP2 plays any role in signaling besides inhibiting the esterase activity. These recent findings indicate the substantial role of /β-hydrolase superfamily proteins in metabolism and signaling of small molecules such as plant hormones.
The site of action of the branch-inhibiting hormone to suppress outgrowth of axillary buds is yet to be determined. Analysis using a genetic mosaic technique demonstrated that MAX2, a putative strigolactone signaling component, works in either the bud or surrounding tissues to sup-press bud outgrowth (Stirnberg et al. 2007). Consistently, MAX2:GUS expression was observed in the whole region of axillary buds. In this study, we showed that D14 mRNA is relatively highly expressed in axillary buds. A higher level of D14 mRNA accumulation in the first leaf bud, which is to be dormant, than in the second leaf bud, which will grow, suggests that D14 may be a limiting factor for the repression of bud outgrowth.
On further investigation of cell specificity of D14 expression by using GUS as a reporter, the D14 promoter was active in xylem parenchyma cells in leaves, stems and axillary buds. As we previously reported, D10:GUS was also expressed in xylem parenchyma cells (Arite et al. 2007). However, in contrast to the D10 promoter which is active in both roots and stems, we were unable to detect D14:GUS activity in roots. Because D10 catalyzes an early step of strigolactone synthesis, expression of D10 in xylem parenchyma cells in roots and stems implies the possibility that strigolactones are synthesized in cells surrounding the xylem and loaded into the xylem to be transported upward. Consistent with this idea, MAX1:GUS is also expressed in the cambial region and xylem-associated parenchyma cells (Booker et al. 2005). We showed that the promoter of D14, which works after the synthesis of strigolactones, is active in xylem parenchyma cells in axillary buds. This suggests that strigolactones transported through the xylem are unloaded and further processed or perceived by D14.
Our phylogenetic analysis shows that D14 homologs are classified into two groups, namely D14 and D14-like subfamilies. It is interesting to note that both D14 and D14-like subfamilies exist in angiosperm species, while only members of the D14-like subfamily have so far been found in non-angiosperm plants. Although we need further analysis by using a larger number of D14 homologs from a variety of plant species to provide more conclusive interpretations, these observations may suggest that the D14 subfamily is confined to angiosperms and that it may have evolved to perform a specialized function that is unique to angiosperms. The origin of symbiosis and shoot branching is ancient (Bonfante and Genre 2008). Arbuscular mycorrhizal fungi form symbiosis with gymnosperms, ferns and bryophytes, suggesting that these non-angiosperm plants also produce strigolactones. In fact, the presence of genes encoding putative CCD7 and CCD8 in P. patens suggests that strigolactone synthesis occurs widely in plants, including non-vascular plants (Gomez-Roldan et al. 2008). Elucidation of the function of D14 homologs in both angiosperm and non-angiosperm plants may reveal how the roles of the two D14 subfamilies evolutionarily diverged and whether the D14-like subfamily also plays a role in strigolactone metabolism or signaling.
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Materials and Methods |
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Plant materials
d3-1, d10-1 and d14-1 mutants (cv. Shiokari) were described previously (Ishikawa et al. 2005
). Plants were grown in a growth chamber (14 h of darkness at 28°C, 10 h of light at 24°C) or in a glasshouse under natural conditions.
Strigolactone analysis
Strigolactone treatment, strigolactone analysis by LC-MS/MS and S. hermonthica germination assays were carried out according to Umehara et al. (2008).
Strigolactone treatment and analysis
Strigolactone treatment and LC/MS-MS analysis of epi-5DS were performed as described previously (Umehara et al. 2008). In brief, surface-sterilized rice seeds were incubated in sterile water in the dark for 2 d. The germinated seeds were transferred into hydroponic culture medium (Kamachi et al. 1991) solidified with 0.6% agar and cultured for 5 d. Each seedling was then transferred to a glass vial containing a sterilized hydroponic culture solution and grown for an additional 7 d (total 14 d). For the phosphate depletion experiment, phosphate ion was omitted from the medium during the hydroponic culture for 7 d. epi-5DS levels in roots and hydroponic culture media (for root exudates) were determined by LC/MS-MS using deuterium-labeled epi-5DS as an internal standard. Strigolactone treatment was carried out by including (+)-strigol or GR24 in hydroponic solution containing phosphate ion for 7 d. Striga hermonthica germination assays were performed as described previously (Sugimoto and Ueyama 2008). Rice seedlings were grown hydroponically in phosphate-depleted medium as described above and the collected medium was extracted with ethyl acetate. Striga hermonthica seeds were pre-conditioned on moist glass fiber filter paper at 30°C for 12 d, and then incubated with the ethyl acetate fraction for 24 h at 30°C before scoring for germination. De-ionized water and (+)-strigol solution were used as negative and positive controls, respectively.
Mapping of D14
To map the D14 locus, the d14-1 mutant was crossed with a wild-type plant (cv. Kasalath). Rough mapping was performed with simple sequence repeat (SSR) markers (M1–M4 and RM232) using 17 mutant plants obtained in the F2 population. Sixty-five F2 mutant plants were used for fine mapping. An SSR marker (IM1) and four derived cleaved amplified polymorphic sequence (dCAPS) markers (IM–IM5) generated based on single nucleotide polymorphisms (SNPs) were used for the fine mapping. Primer sets used for mapping are 5'-AATCCGGTTGAGGTTGACAC-3' and 5'-ATTTCAGTTCGGCGAGAGG-3' (M1), 5'-GAATGCACTTAGGGTCAAAAG-3' and 5'-AACTTGGCGTGGCACATATT-3' (M2), 5'-TGAGTTAAACCCCTGAAAACAG-3' and 5'-CCCATTGATTGTTCTCGAAAG-3' (M3), 5'-AGAGCGAAACCCTAGGCAAC-3' and 5'-CAATCCTAGCTATAACCTGGACA-3' (M4), 5'-CCGGTATCCTTCGATATTGC-3' and 5'-CCGACTTTTCCTCCTGACG-3' (RM232), 5'-GATGTCGCGAAACAAAATCC-3' and 5'-CGTCCGTAGCTAAAAGACTG-3' (IM1), 5'-CCCAATATTTCAGTTAGTGGTGA-3' and 5'-GCTAGGAGAATTGGGGGTTT-3' (IM2), 5'-CAGCCGGACTAAAATCTTTCTT-3' and 5'-CTGCTGCTCGGGCTACTG-3' (IM3), 5'-TTCAAACTCGACGTGTGGAC-3' and 5'-CGTTCGGTCTTTTTGGTCTC-3' (IM4) and 5'-TCGCTAGCTACTTTTCTGATGCGGATGCATG-3' and 5'-TGTGTTTTGTCCGATTGACC-3' (IM5).
Complementation of the d14-1 phenotype
BAC DNA (OSJNBa0009J13) was digested with BamHI and XbaI, and a 4.9 kb DNA fragment containing the entire putative D14 gene was cloned into pPZP (Hajdukiewicz et al. 1994). The resultant binary vector was introduced into d14-1 via Agrobacterium-mediated transformation.
RT–PCR
RT–PCR was performed according to Arite et al. (2007). PCR was performed using the following primer sets: D14, 5'-GTGCTGTCGCATGGCTTC-3' and 5'- GCAGGTCGTCGACGTAGG-3'; and Actin, 5'-CAATCGTGAGAAGATGACCC-3' and 5'-GTCCATCAGGAAGCTCGTAGC-3'.
Analysis of D14 expression pattern
To construct pD14:GUS, approximately 2.7 kb of the D14 promoter region was amplified by PCR with a set of primers (5'-GGATCCCCTTGTCTAAGACC-3' and 5'-CACACCAGCGCGGCGGATTG-3') using BAC DNA (OSJNBa0009J13) as a template. The amplified product was cloned into pENTR1A (Invitrogen, Calsbad, CA, USA) and subsequently cloned into pGWB3 (Nakagawa et al. 2007) with LR clonase (Invitrogen). The resultant binary vector was introduced into Nipponbare via Agrobacterium-mediated transformation. Regenerated T0 transgenic plants were used for histochemical staining of GUS activity. Leaves and stems were cut by hand, treated with 90% acetone for 15 min on ice and stained with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) solution containing 0.5 mg ml–1 X-gluc, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.1% Triton X-100, 10 mM EDTA and 100 mM NaPO4 (pH 7.0). Samples were deaerated at the initiation of staining with X-gluc solution.
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Supplementary data |
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Supplementary data are available at PCP online.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary dataFundingAcknowledgmentsReferences |
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The Ministry of Agriculture, Forestry and Fisheries (Genomics for Agricultural Innovation, IPG-0001 to J.K.); the Ministry of Education, Culture, Sports, Science and Technologies (Grant-in-Aid for Scientific Research on Priority Areas to J.K.; RIKEN President’s Discretionary Fund (Strategic Programs for R&D to S.Y.); RIKEN Special Postdoctoral Researchers Program (to M.U.).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary dataFundingAcknowledgmentsReferences |
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We thank Kohki Akiyama (Osaka Prefecture University) and Koichi Yoneyama (Utsunomiya University) for providing epi-5DS and GR24, respectively, Takayuki Kochi and Katsuyuki Yamato (Kyoto University) for information on Marchantia polymorpha expressed sequence tag sequences, Tsuyoshi Nakagawa (Shimane University) for pGWB3, Hiromu Kameoka for GUS staining, and Miho Takemura and Kanji Ohyama for providing T.A. with facilities for carrying out the experiments. We thank Kaoru Fujiwara (RIKEN) for assistance in shoot branching assays.
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References |
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Akiyama K, Matsuzaki K, Hayashi H. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature (2005) 435:824–827.[CrossRef][Medline]
Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M, et al. DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J. (2007) 51:1019–1029.[CrossRef][Web of Science][Medline]
Beveridge CA. Axillary bud outgrowth: sending a message. Curr. Opin. Plant Biol. (2006) 9:35–40.[CrossRef][Web of Science][Medline]
Bonfante P, Genre A. Plants and arbuscular mycorrhizal fungi: an evolutionary–developmental perspective. Trends Plant Sci. (2008) 13:492–498.[CrossRef][Web of Science][Medline]
Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O. MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Curr. Biol. (2004) 14:1232–1238.[CrossRef][Web of Science][Medline]
Booker J, Sieberer T, Wright W, Williamson L, Willett B, Stirnberg P, et al. MAX1 encodes a cytochrome P450 family member that acts downstream of MAX3/4 to produce a carotenoid-derived branch-inhibiting hormone. Dev. Cell (2005) 8:443–449.[CrossRef][Web of Science][Medline]
Brody MS, Vijay K, Price CW. Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis. J. Bacteriol. (2001) 183:6422–6428.
Cook CE, Whichard LP, Wall ME, Egley GH, Coggon P, Luhan PA, et al. Germination stimulants II. The structure of strigol—a potent seed germination stimulant for witchweed (Striga lutea Lour). J. Amer. Chem. Soc. (1972) 94:6198–6199.[CrossRef][Web of Science]
Foo E, Bullier E, Goussot M, Foucher F, Rameau C, Beveridge CA. The branching gene RAMOSUS1 mediates interactions among two novel signals and auxin in pea. Plant Cell (2005) 17:464–474.
Forouhar F, Lee IS, Vujcic J, Vujcic S, Shen J, Vorobiev SM, et al. Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proc. Natl Acad. Sci. USA (2005) 102:1773–1778.
Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP, et al. Strigolactone inhibition of shoot branching. Nature (2008) 455:189–94.[CrossRef][Web of Science][Medline]
Hajdukiewicz P, Svab Z, Maliga P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transforma-tion. Plant Mol. Biol. (1994) 25:989–994.[CrossRef][Web of Science][Medline]
Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J. Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol. (2005) 46:79–86.
Jhonson X, Brcich T, Dun EA, Goussot M, Haurogné K, Beveridge CA, et al. Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals. Plant Physiol. (2006) 142:1014–1026.
Kamachi K, Yamaya T, Mae T, Ojima K. A role for glutamine synthetase in the recombination of leaf nitrogen during natural senescence in rice leaves. Plant Physiol. (1991) 96:411–417.
Kaneko T, Tanaka N, Kumasaka T. Crystal structures of RsbQ, a stress-response regulator in Bacillus subtilis. Protein Sci. (2005) 14:558–565.[CrossRef][Web of Science][Medline]
Kumar D, Klessig D. High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity. Proc. Natl Acad. Sci. USA (2003) 100:16101–16106.
McSteen P, Leyser O. Shoot branching. Annu. Rev. Plant Biol. (2005) 56:353–374.[CrossRef][Medline]
Murase K, Hirano YSun, Hakoshima T, T. Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature (2008) 456:459–464.[CrossRef][Web of Science][Medline]
Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, et al. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Biosci. Biotechnol. Biochem. (2007) 71:2095–2100.[CrossRef][Medline]
Nardini M, Dijkstra BW. Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. (1999) 9:732–737.[CrossRef][Web of Science][Medline]
Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, et al. The alpha/beta hydrolase fold. Protein Eng. (1992) 5:197–211.
Ongaro V, Leyser O. Hormonal control of shoot branching. J. Exp. Bot. (2008) 59:67–74.
Shimada A, Ueguchi-Tanaka M, Nakatsu T, Nakajima M, Naoe Y, Ohmiya H, et al. Structural basis for gibberellin recognition by its receptor GID1. Nature (2008) 456:520–524.[CrossRef][Web of Science][Medline]
Snowden KC, Simkin AJ, Janssen BJ, Templeton KR, Loucas HM, Simons JL, et al. The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development. Plant Cell (2005) 17:746–759.
Sorefan K, Booker J, Haurogné K, Goussot M, Bainbridge K, Foo E, et al. MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea. Genes Dev. (2003) 17:1469–1474.
Steeves TA, Sussex IM. Patterns in Plant Development. (1989) 2nd edn. Cambridge, UK: Cambridge University Press.
Stirnberg P, Furner IJ, Leyser H.MO. MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching. Plant J. (2007) 50:80–94.[CrossRef][Web of Science][Medline]
Stirnberg P, van de Sande K, Leyser H.MO. MAX1 and MAX2 control shoot lateral branching in Arabidopsis. Development (2002) 129:1131–1141.
Sugimoto Y, Ueyama T. Production of (+)-5-deoxystrigol by Lotus japonicus root culture. Phytochemistry (2008) 69:212–217.[CrossRef][Web of Science][Medline]
Tsugane K, Maekawa M, Takagi K, Takahara H, Qian Q, Eun CH, et al. An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice. Plant J. (2006) 45:46–57.[CrossRef][Web of Science][Medline]
Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh H, Katoh E, Kobayashi M, et al. GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberelin. Nature (2005) 437:693–698.[CrossRef][Medline]
Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, et al. Inhibition of shoot branching by new terpenoid plant hormones. Nature (2008) 455:195–200.[CrossRef][Web of Science][Medline]
Zou J, Zhang S, Zhang W, Li G, Chen Z, Zhai W, et al. The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. Plant J. (2006) 48:687–698.[CrossRef][Web of Science][Medline]
(Received May 14, 2009; Accepted June 16, 2009)
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