DWARF27 Research Articles

The Psathyrostachys juncea DWARF27 gene encodes an all-trans-/9-cis-beta-carotene isomerase in the control of plant branches in Arabidopsis thaliana by strigolactones.

Strigolactones (SLs), carotenoid-derived plant hormones, govern the growth and development of both monocotyledonous and dicotyledonous plants. DWARF27 (D27), a plastid-targeted protein located at the initiation site of the core pathway in SL synthesis, plays a crucial role in regulating plant tillering (branching). In rice (Oryza sativa) and wheat (Triticum aestivum), OsD27 and TaD27-B proteins modulate the number of plant tillers by participating in SL biosynthesis. Similarly, AtD27 in Arabidopsis thaliana is required for SL production and has a significant impact on phenotypic changes related to branching. At the same time, TaD27 in wheat has been confirmed as a functional orthologue of AtD27 in Arabidopsis, and both Psathyrostachys juncea and wheat belong to the Triticeae, so we speculate that PjD27 gene may also have the same function as AtD27 in Arabidopsis. In this study, we initially screened the PjD27 gene significantly associated with tillering regulation through transcriptome data analysis and subsequently validated its expression levels using qRT-PCR analysis. Furthermore, we conducted phylogenetic analysis using amino acid sequences from 41 species, including P. juncea, to identify closely related species of P. juncea. Here, we analyze the conservation of D27 protein among P. juncea, rice, wheat, and Arabidopsis and provide preliminary evidence suggesting that PjD27 protein is an orthologue of D27 protein in Arabidopsis. Through reverse genetics, we demonstrate the crucial role of PjD27 in regulating plant branching, establishing it as a functional orthologue of D27 in Arabidopsis. Furthermore, following transient expression in tobacco (Nicotiana tabacum), we demonstrate that the subcellular location of the PjD27 protein is consistent with the cellular location of TaD27-B in wheat. Quantitative analysis of SLs shows that PjD27 is a key gene regulating tillering (branching) by participating in SL biosynthesis. By elucidating the function of the PjD27 gene, our findings provide valuable genetic resources for new germplasm creation and improving grain yield in P. juncea.

DWARF27 and CAROTENOID CLEAVAGE DIOXYGENASE 7 genes regulate release, germination and growth of gemma in Marchantia polymorpha.

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.

Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-β-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-β-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-β-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-β-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-β-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

D27-LIKE1 isomerase has a preference towards trans/cis and cis/cis conversions of carotenoids in Arabidopsis.

Carotenoids contribute to a variety of physiological processes in plants, functioning also as biosynthesis precursors of ABA and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-β-carotene to 9-cis-β-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2, which were predicted to be β-carotene-isomerases. In the present study, we characterised D27-LIKE1 and identified some key aspects of its physiological and enzymatic functions in Arabidopsis. d27-like1-1 mutant does not display any strigolactone-deficient traits and exhibits a substantially higher 9-cis-violaxanthin content, which is accompanied by a slightly higher ABA level. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all β-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-β-carotene forms, and accepts zeaxanthin and violaxanthin as substrates. Finally, we present evidence showing that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with a long evolutionary history. In summary, we demonstrate that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and, eventually, SL and ABA biosynthesis pathways.

Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum).

Strigolactones (SLs) are a class of phytohormones that regulate plant shoot branching and adventitious root development. However, little is known regarding the role of SLs in controlling the behavior of the smallest unit of the organism, the single cell. Here, taking advantage of a classic single-cell model offered by the cotton (Gossypium hirsutum) fiber cell, we show that SLs, whose biosynthesis is fine-tuned by gibberellins (GAs), positively regulate cell elongation and cell wall thickness by promoting the biosynthesis of very long-chain fatty acids (VLCFAs) and cellulose, respectively. Furthermore, we identified two layers of transcription factors (TFs) involved in the hierarchical regulation of this GA-SL crosstalk. The top-layer TF GROWTH-REGULATING FACTOR 4 (GhGRF4) directly activates expression of the SL biosynthetic gene DWARF27 (D27) to increase SL accumulation in fiber cells and GAs induce GhGRF4 expression. SLs induce the expression of four second-layer TF genes (GhNAC100-2, GhBLH51, GhGT2, and GhB9SHZ1), which transmit SL signals downstream to two ketoacyl-CoA synthase genes (KCS) and three cellulose synthase (CesA) genes by directly activating their transcription. Finally, the KCS and CesA enzymes catalyze the biosynthesis of VLCFAs and cellulose, respectively, to regulate development of high-grade cotton fibers. In addition to providing a theoretical basis for cotton fiber improvement, our results shed light on SL signaling in plant development at the single-cell level.

Genome wide analysis of DWARF27 genes in soybean and functional characterization of GmD27c reveals eminent role of strigolactones in rhizobia interaction and nodulation in Glycine max.

Strigolactones (SLs) are newly identified hormones and their biosynthesis is stimulated under phosphate deprivation and accomplished by the action of several enzymes, including the beta-carotene isomerase DWARF27 (D27). Expression of D27 is well renowned to respond to phosphate insufficiency. However, the identification and functional analysis of the carotenoid isomerase D27 genes are not elucidated in soybean. A total of six D27 genes were identified in the soybean genome and designated on the basis of chromosomal localization. According to the findings, these genes were irregularly distributed on chromosomes, and segmental repetition led to the expansion of the soybean GmD27 gene family. Based on a neighbor-joining phylogenetic tree, the predicted D27 proteins of soybean were divided into three clades. Based on RNA seq data analysis, GmD27 genes were differently expressed in various tissues but GmD27c was the highest. Therefore, GmD27c was chosen for the additional functional study due to its rather obvious transcription in nodulation and roots. RT-qPCR results showed that GmD27c was highly expressed in different nodule stages and in response to rhizobia infection. Functional characterization of GmD27c revealed that overexpression of GmD27c led to higher nodule number, while GmD27c knockdown caused fewer nodules compared to GUS control. Furthermore, GmD27c overexpressed and knockdown lines oppositely regulated the expression of numerous nodulation genes, which are vital for the development of nodules. This study not only discovered that SL biosynthesis and signaling pathway genes are conserved, but it also revealed that SL biosynthesis gene GmD27c and legume rhizobia have close interactions in controlling plant nodule number.

Comparative transcriptome analyses reveal different mechanism of high- and low-tillering genotypes controlling tiller growth in orchardgrass (Dactylis glomerata L.)

BackgroundTillering is an important agronomic trait underlying the yields and reproduction of orchardgrass (Dactylis glomerata), an important perennial forage grass. Although some genes affecting tiller initiation have been identified, the tillering regulatory network is still largely unknown, especially in perennial forage grasses. Thus, unraveling the regulatory mechanisms of tillering in orchardgrass could be helpful in developing selective strategies for high-yield perennial grasses. In this study, we generated high-throughput RNA-sequencing data from multiple tissues of tillering stage plants to identify differentially expressed genes (DEGs) between high- and low-tillering orchardgrass genotypes. Gene Ontology and pathway enrichment analyses connecting the DEGs to tillering number diversity were conducted.ResultsIn the present study, approximately 26,282 DEGs were identified between two orchardgrass genotypes, AKZ-NRGR667 (a high-tillering genotype) and D20170203 (a low-tillering genotype), which significantly differed in tiller number. Pathway enrichment analysis indicated that DEGs related to the biosynthesis of three classes of phytohormones, i.e., strigolactones (SLs), abscisic acid (ABA), and gibberellic acid (GA), as well as nitrogen metabolism dominated such differences between the high- and low-tillering genotypes. We also confirmed that under phosphorus deficiency, the expression level of the major SL biosynthesis genes encoding DWARF27 (D27), 9-cis-beta-carotene 9′,10′-cleaving dioxygenase (CCD7), carlactone synthase (CCD8), and more axillary branching1 (MAX1) proteins in the high-tillering orchardgrass genotype increased more slowly relative to the low-tillering genotype.ConclusionsHere, we used transcriptomic data to study the tillering mechanism of perennial forage grasses. We demonstrated that differential expression patterns of genes involved in SL, ABA, and GA biosynthesis may differentiate high- and low-tillering orchardgrass genotypes at the tillering stage. Furthermore, the core SL biosynthesis-associated genes in high-tillering orchardgrass were more insensitive than the low-tillering genotype to phosphorus deficiency which can lead to increases in SL biosynthesis, raising the possibility that there may be distinct SL biosynthesis way in tillering regulation in orchardgrass. Our research has revealed some candidate genes involved in the regulation of tillering in perennial grasses that is available for establishment of new breeding resources for high-yield perennial grasses and will serve as a new resource for future studies into molecular mechanism of tillering regulation in orchardgrass.

Broomrape infestation in carrot (Daucus carota): Changes in carotenoid gene expression and carotenoid accumulation in the parasitic weed Phelipanche aegyptiaca and its host

Carotenogenesis has been intensively studied in carrot roots, and transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about the transcriptional regulation of carotenoid biosynthetic genes concerning carotenoid accumulation during infestation by the obligate parasite Phelipanche aegyptiaca. HPLC analysis revealed a decrease in carotenoid levels of the different carrot cultivars when parasitized by P. aegyptiaca. Besides, we isolated and analyzed P. aegyptiaca tubercles parasitizing the various carrot root cultivars and show that they accumulate different carotenoids compared to those in non-infested carrot roots. Expression analysis of PHYTOENE SYNTHASE (PSY1) and CAROTENOID ISOMERASE (CRTISO) as well as the strigolactone apocarotenoid biosynthetic genes DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 revealed that their transcript levels showed significant variation in P. aegyptiaca infested carrot roots. After parasite infestation, the expression of these genes was strongly reduced, as were the carotenoid levels and this was more pronounced in the uncommon non-orange varieties. We also analyzed the parasite genes encoding D27, CCD7 and CCD8 and show that they are expressed in tubercles. This raises important questions of whether the parasite produces its carotenoids and apocarotenoids including strigolactones and whether the latter might have a role in tubercle development.

Effect of D-ring C-3’ methylation of strigolactone analogs on their transcription regulating activity in rice

ABSTRACTStrigolactones (SLs) are a well-known class of plant hormones, which are involved in a number of developmental and adaptation processes and mediate different interspecific interactions. In spite of the growing knowledge on SL biosynthesis and signal transduction, effects of structural modifications on the activity and efficiency of SLs and their analogs remain largely elusive. SLs are characterized by the presence of a lactone ring (D-ring) that is connected by an enol ether bridge to a second moiety. In this study, we investigated the effect of additional D-ring methylation of SL analogs on their transcription regulating activity. For this purpose, we compared the SL analogs MP13 and AR8, which differ only by the presence of a methyl group at the C-3ʹ atom in the latter. Transcription regulating activity was determined by quantitative real-time PCR measurement of transcript levels of SL-dependent, feed-back regulated genes in treated wild type and ccd7 mutant rice seedlings. Results obtained indicate that C-3ʹ methylation reduces the transcription regulating activity, as shown by the more pronounced suppression of the SL biosynthesis genes DWARF27 (D27) and CAROTENOID CLEAVAGE DIOXYGENASES (CCD7 and CCD8) and higher induction of the SL signaling repressor gene DWARF53 (D53) in MP13 treated seedlings. These results are consistent with a recent study on the biological activities of MP13 and AR8.

Strigolactone-Based Node-to-Bud Signaling May Restrain Shoot Branching in Hybrid Aspen.

The biosynthesis and roles of strigolactones (SLs) have been investigated in herbaceous plants, but so far, their role in trees has received little attention. In this study, we analyzed the presence, spatial/temporal expression and role of SL pathway genes in Populus tremula � Populus tremuloides. In this proleptic species, axillary buds (AXBs) become para-dormant at the bud maturation point, providing an unambiguous starting point to study AXB activation. We identified previously undescribed Populus homologs of DWARF27 (D27), LATERAL BRANCHING OXIDOREDUCTASE (LBO) and DWARF53-like (D53-like) and analyzed the relative expression of all SL pathway genes in root tips and shoot tissues. We found that, although AXBs expressed MORE AXILLARY GROWTH1 (MAX1) and LBO, they did not express MAX3 and MAX4, whereas nodal bark expressed high levels of all SL biosynthesis genes. By contrast, expression of the SL perception and signaling genes MAX2, D14 and D53 was high in AXBs relative to nodal bark and roots. This suggests that AXBs are reliant on the associated nodes for the import of SLs and SL precursors. Activation of AXBs was initiated by decapitation and single-node isolation. This rapidly downregulated SL pathway genes downstream of MAX4, although later these genes were upregulated coincidently with primordia formation. GR24-feeding counteracted all activation-related changes in SL gene expression but did not prevent AXB outgrowth showing that SL is ineffective once AXBs are activated. The results indicate that nodes rather than roots supply SLs and its precursors to AXBs, and that SLs may restrain embryonic shoot elongation during AXB formation and para-dormancy in intact plants.

The Arabidopsis DWARF27 gene encodes an all-trans-/9-cis-β-carotene isomerase and is induced by auxin, abscisic acid and phosphate deficiency

Strigolactones (SLs) are carotenoid-derived plant hormones that influence various aspects of plant growth and development in response to environmental conditions, especially nutrients deficiency. SLs are synthesized via a strict stereo-specific core pathway that leads to the intermediate carlactone, requiring the iron-containing polypeptide DWARF27 (D27) and the carotenoid cleavage dioxygenases 7 (CCD7) and 8 (CCD8). It has been shown that the rice OsD27 is a β-carotene isomerase catalyzing the interconversion of all-trans- into 9-cis-β -carotene. However, data about the enzymatic activity of D27 from other species are missing. Here, we investigated the activity and substrate specificity of the Arabidopsis AtD27 by testing a broad range of carotenoid substrates. Both in vivo and in vitro assays show that AtD27 catalyzes the reverse isomerization of all-trans-/9-cis-β-carotene. AtD27 did not isomerize 13-cis- or 15-cis-β-carotene, indicating high specificity for the C9-C10 double bond. The isomerization reaction was inhibited in the presence of silver acetate, pointing to the involvement of an iron-sulfur cluster. We further investigated the expression of AtD27, using Arabidopsis transgenic lines expressing β-glucuronidase (GUS) under the control of AtD27 native promoter. AtD27 is ubiquitously expressed throughout the plant with the highest expression in immature flowers. In lateral roots, AtD27 expression was induced by treatment with auxin and ABA, while the application of SL analogs did not show an effect. Lower ABA levels in atd27 mutant indicated an interference with the ABA pathway. Quantitative real-time RT-PCR showed that transcript levels of AtD27 and other SL biosynthetic genes in roots are induced upon phosphate starvation. Taken together, our study on AtD27 confirms the postulated enzymatic function of this enzyme, shows its strict substrate- and regio-specificity and indicates an important role in response to multiple plant hormones and phosphate deficiency.

Upregulation of DWARF27 is associated with increased strigolactone levels under sulfur deficiency in rice.

Plants produce strigolactones (SLs) in roots in response to nitrogen or phosphate deficiency. To evaluate SL levels under other mineral deficiencies in rice, we cultivated rice seedlings in hydroponic media without nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, and iron. Tiller bud outgrowth was stimulated under calcium deficiency because of low SL levels. SL levels increased under sulfur deficiency, in addition to phosphate, and nitrogen deficiencies. To explore which genes are key regulators of SL production under sulfur deficiency, we analyzed the expression of SL‐related genes in sulfur‐sufficient and sulfur‐deficient conditions. An SL biosynthesis gene, DWARF27 (D27), was strongly expressed under sulfur deficiency, and its expression was decreased by sulfur supply. The levels of D10, D17, and OsMAX1 transcripts did not differ between sulfur‐sufficient and sulfur‐deficient conditions. These results suggest that the increased SL levels under sulfur deficiency are due to a high expression of D27. A combination of nitrogen, phosphorus, and sulfur deficiencies had no additive synergistic effect on SL production. Under combined phosphorus and sulfur deficiency, the expression levels of most SL biosynthesis genes were elevated. The number of tiller buds in the d27 mutant was higher than in the wild type, but lower than in other d mutants. Under sulfur deficiency, the chlorophyll content of d27 was lower than those of other d mutants. These results indicate that D27 plays an important role in adaptation to sulfur deficiency in rice.

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis

BackgroundStrigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis.ResultsWe provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2.ConclusionOur data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0651-x) contains supplementary material, which is available to authorized users.

Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.

Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro.

Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.