Increase in ENHANCER OF SHOOT REGENERATION2 expression by treatment with strigolactone-related inhibitors and kinetin during adventitious shoot formation in ipecac.

KEY MESSAGE: Increase of ENHANCER OF SHOOT REGENERATION 2 expression was consistent to treatment with kinetin, TIS108, and KK094 in adventitious shoot formation of ipecac. Unlike many plant species, ipecac (Carapichea ipecacuanha (Brot.) L. Andersson) can form adventitious shoots in tissue culture without cytokinin (CK) treatment. Strigolactone (SL) biosynthesis and signaling inhibitors stimulate adventitious shoot formation in ipecac, suggesting their potential use as novel growth regulators in plant tissue culture, but the molecular mechanism of their action is unclear. In this study, we compared the effects of SL-related inhibitors (TIS108 and KK094) and CKs (2iP, tZ, and kinetin) on adventitious shoot formation in ipecac. Exogenously applied SL-related inhibitors and CKs stimulated adventitious shoot formation. Combinations of SL-related inhibitors and kinetin also promoted adventitious shoot formation, but without additive effects. We also analyzed the expression of CK biosynthesis genes in ipecac. TIS108 increased the expression of the ipecac homolog of ISOPENTENYL TRANSFERASE 3 (CiIPT3) but decreased that of LONELY GUY 7 homolog (CiLOG7), presumably resulting in no change in 2iP-type CK levels. KK094 and kinetin increased CiLOG7 expression, elevating 2iP-type CK levels. Among pluripotency- and meristem-related genes, TIS108, KK094, and kinetin consistently increased the expression of ENHANCER OF SHOOT REGENERATION 2 homolog (CiESR2), which has a key role in shoot regeneration, in the internodal segment region that formed adventitious shoots. We propose that CiESR2 might be a key stimulator of adventitious shoot formation in ipecac.

Gene expression profiling before and after internode culture for adventitious shoot formation in ipecac.

BACKGROUND: In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), adventitious shoots can be induced simply by placing internodal segments on phytohormone-free culture medium. The shoots form locally on the epidermis of the apical region of the segments, but not the basal region. Levels of endogenous auxin and cytokinin transiently increase in the segments after 1 week of culture. RESULTS: Here, we conducted RNA-seq analysis to compare gene expression patterns in apical and basal regions of segments before culture and after 1 week of culture for adventitious shoot formation. The results revealed 8987 differentially expressed genes in a de novo assembly of 76,684 genes. Among them, 276 genes were upregulated in the apical region after 1 week of culture relative to before culture and the basal region after 1 week of culture. These genes include 18 phytohormone-response genes and shoot-formation-related genes. Validation of the gene expression by quantitative real-time PCR assay confirmed that the expression patterns were similar to those of the RNA-seq data. CONCLUSIONS: The transcriptome data show that expression of cytokinin biosynthesis genes is induced along with the acquisition of cellular pluripotency and the initiation of cell division by wounding in the apical region of internodal segments, that trigger adventitious shoot formation without callusing.

Strigolactone signaling inhibition increases adventitious shoot formation on internodal segments of ipecac.

MAIN CONCLUSION: SL inhibited adventitious shoot formation of ipecac, whereas the SL-related inhibitors promoted adventitious shoot formation. SL-related inhibitors might be useful as new plant growth regulators for plant propagation. In most plant species, phytohormones are required to induce adventitious shoots for propagating economically important crops and regenerating transgenic plants. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Here we evaluated the effects of GR24 (a synthetic strigolactone, SL), SL biosynthetic inhibitors, and an SL antagonist on adventitious shoot formation during tissue culture of ipecac. We found that exogenously applied GR24 suppressed indole-3-acetic acid transport in internodal segments and decreased the number of adventitious shoots formed; in addition, the distribution of adventitious shoots changed from the apical to middle region of the internodal segments. In contrast, the SL-related inhibitors promoted adventitious shoot formation on both apical and middle regions of the segments. In particular, SL antagonist treatment increased endogenous cytokinin levels and induced multiple shoot development. These results indicate that SL inhibits adventitious shoot formation in ipecac. In ipecac, one of the shoots in each internodal segment becomes dominant and auxin derived from that shoot suppresses the other shoot growth. Here, this dominance was overcome by application of SL-related inhibitors. Therefore, SL-related inhibitors might be useful as new plant growth regulators to improve the efficiency of plant propagation in vitro.

Endogenous auxin determines the pattern of adventitious shoot formation on internodal segments of ipecac.

MAIN CONCLUSION: Endogenous auxin determines the pattern of adventitious shoot formation. Auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots. Adventitious shoot formation is required for the propagation of economically important crops and for the regeneration of transgenic plants. In most plant species, phytohormones are added to culture medium to induce adventitious shoots. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Thus, ipecac culture allows us to investigate the effects of endogenous phytohormones during adventitious shoot formation. In phytohormone-free culture, adventitious shoots were formed on the apical region of the internodal segments, and a high concentration of IAA was detected in the basal region. To explore the relationship between endogenous auxin and adventitious shoot formation, we evaluated the effects of auxin transport inhibitors, auxin antagonists, and auxin biosynthesis inhibitors on adventitious shoot formation in ipecac. Auxin antagonists and biosynthesis inhibitors strongly suppressed adventitious shoot formation, which was restored by exogenously applied auxin. Auxin biosynthesis and transport inhibitors significantly decreased the IAA level in the basal region and shifted the positions of adventitious shoot formation from the apical region to the middle region of the segments. These data indicate that auxin determines the positions of the shoots formed on internodal segments of ipecac. Only one of the shoots formed grew vigorously; this phenomenon is similar to apical dominance. When the largest shoot was cut off, other shoots started to grow. Naphthalene-1-acetic acid treatment of the cut surface suppressed shoot growth, indicating that auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots.

Regulation of Strigolactone Biosynthesis by Gibberellin Signaling.

Strigolactones (SLs) are a class of plant hormones that regulate diverse physiological processes, including shoot branching and root development. They also act as rhizosphere signaling molecules to stimulate the germination of root parasitic weeds and the branching of arbuscular mycorrhizal fungi. Although various types of cross talk between SLs and other hormones have been reported in physiological analyses, the cross talk between gibberellin (GA) and SLs is poorly understood. We screened for chemicals that regulate the level of SLs in rice (Oryza sativa) and identified GA as, to our knowledge, a novel SL-regulating molecule. The regulation of SL biosynthesis by GA is dependent on the GA receptor GID1 and F-box protein GID2. GA treatment also reduced the infection of rice plants by the parasitic plant witchers weed (Striga hermonthica). These data not only demonstrate, to our knowledge, the novel plant hormone cross talk between SL and GA, but also suggest that GA can be used to control parasitic weed infections.

Low Infection of Phelipanche aegyptiaca in Micro-Tom Mutants Deficient in CAROTENOIDCLEAVAGE DIOXYGENASE 8.

Strigolactones (SLs), a group of plant hormones, induce germination of root-parasitic plants and inhibit shoot branching in many plants. Shoot branching is an important trait that affects the number and quality of flowers and fruits. Root-parasitic plants, such as Phelipanche spp., infect tomato roots and cause economic damage in Europe and North Africa-hence why resistant tomato cultivars are needed. In this study, we found carotenoid cleavage dioxygenase 8-defective mutants of Micro-Tom tomato (slccd8) by the "targeting induced local lesions in genomes" (TILLING) method. The mutants showed excess branching, which was suppressed by exogenously applied SL. Grafting shoot scions of the slccd8 mutants onto wild-type (WT) rootstocks restored normal branching in the scions. The levels of endogenous orobanchol and solanacol in WT were enough detectable, whereas that in the slccd8 mutants were below the detection limit of quantification analysis. Accordingly, root exudates of the slccd8 mutants hardly stimulated seed germination of root parasitic plants. In addition, SL deficiency did not critically affect the fruit traits of Micro-Tom. Using a rhizotron system, we also found that Phelipanche aegyptiaca infection was lower in the slccd8 mutants than in wild-type Micro-Tom because of the low germination. We propose that the slccd8 mutants might be useful as new tomato lines resistant to P. aegyptiaca.

Carlactone is an endogenous biosynthetic precursor for strigolactones.

Strigolactones (SLs) are a class of terpenoid plant hormones that regulate shoot branching as well as being known as root-derived signals for symbiosis and parasitism. SL has tricyclic-lactone (ABC-ring) and methyl butenolide (D-ring), and they are connected through an enol ether bridge. Recently, a putative biosynthetic intermediate called carlactone (CL), of which carbon skeleton is in part similar to those of SLs, was identified by biochemical analysis of three biosynthetic enzymes, DWARF27, CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), and CCD8 in vitro. However, CL has never been identified from plant tissues, and the conversion of CL to SLs has not been proven in vivo. To address these questions, we chemically synthesized (13)C-labeled CL. We show that (13)C-labeled CL is converted to (-)-[(13)C]-2'-epi-5-deoxystrigol ((-)-2'-epi-5DS) and [(13)C]-orobanchol, endogenous SLs in rice, in the dwarf10 mutant, which is defective in CCD8. In addition, we successfully identified endogenous CL by using liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry in rice and Arabidopsis. Furthermore, we determined the absolute stereochemistry of endogenous CL to be (11R)-configuration, which is the same as that of (-)-2'-epi-5DS at the corresponding position. Feeding experiments showed that only the (11R)-isomer of CL, but not the (11S)-isomer, was converted to (-)-2'-epi-5DS in vivo. Taken together, our data provide conclusive evidence that CL is an endogenous SL precursor that is stereospecifically recognized in the biosynthesis pathway.

Structural Requirements of Strigolactones for Shoot Branching Inhibition in Rice and Arabidopsis.

The structural requirements of strigolactones (SLs) involved in germination induction of root parasitic plants and hyphal branching in arbuscular mycorrhizal (AM) fungi have been extensively studied. However, our knowledge of the requirements of SLs involved in shoot branching inhibition in plants is still limited. To address this question, we investigated the structure-activity relationships of SLs in shoot branching inhibition in rice and Arabidopsis. SLs possess a four-ring structure, with a tricyclic lactone (ABC-rings) connected to a methylbutenolide part (D-ring) via an enol ether bridge. Here, we show that the the (R) configuration at C-2', which determines the steric position of the D-ring relative to the enol ether olefin bond, is critical for the hormonal activity in rice. Replacement of the enol ether moiety by an alkoxy or imino ether resulted in a severe reduction in biological activity in rice. Moreover, yeast two-hybrid experiments using a possible SL receptor, DWARF14 (D14), and a repressor in the SL signaling pathway, DWARF53 (D53), showed that D14 can interact with D53 in the presence of (2'R) stereoisomers of SLs, but not (2'S) stereoisomers, suggesting that the stereostructure of SLs is crucial for the interaction of these proteins. When GR5, an AB-ring-truncated analog, was applied to the hydroponic culture medium, strong inhibition of shoot branching was observed both in rice and in Arabidopsis. However, GR5 was only weakly active when directly applied to the axillary buds of Arabidopsis. Our results indicate that the difference in plant species and application methods greatly influences the apparent SL biological activity.

Strigolactone signaling regulates rice leaf senescence in response to a phosphate deficiency.

Strigolactones (SLs) act as plant hormones that inhibit shoot branching and stimulate secondary growth of the stem, primary root growth, and root hair elongation. In the moss Physcomitrella patens, SLs regulate branching of chloronemata and colony extension. In addition, SL-deficient and SL-insensitive mutants show delayed leaf senescence. To explore the effects of SLs on leaf senescence in rice (Oryza sativa L.), we treated leaf segments of rice dwarf mutants with a synthetic SL analogue, GR24, and evaluated their chlorophyll contents, ion leakage, and expression levels of senescence-associated genes. Exogenously applied GR24 restored normal leaf senescence in SL-deficient mutants, but not in SL-insensitive mutants. Most plants highly produce endogenous SLs in response to phosphate deficiency. Thus, we evaluated effects of GR24 under phosphate deficiency. Chlorophyll levels did not differ of in the wild-type between the sufficient and deficient phosphate conditions, but increased in the SL-deficient mutants under phosphate deficiency, leading in the strong promotion of leaf senescence by GR24 treatment. These results indicate that the mutants exhibited increased responsiveness to GR24 under phosphate deficiency. In addition, GR24 accelerated leaf senescence in the intact SL-deficient mutants under phosphate deficiency as well as dark-induced leaf senescence. The effects of GR24 were stronger in d10 compared to d17. Based on these results, we suggest that SLs regulate leaf senescence in response to phosphate deficiency.

Inhibition of shoot branching by new terpenoid plant hormones.

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class-or their biosynthetic precursors-in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.

The D3 F-box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis.

Shoot has important roles in strigolactone production of rice roots under sulfur deficiency.

Strigolactones (SLs) are a class of plant hormones that control plant architecture. SL levels in roots are determined by the nutrient conditions in the rhizosphere, especially the levels of nitrogen (N) and phosphorus (P). Our previous research showed that SL production is induced in response to deficiency of sulfur (S) as well as of N and P, and inhibits shoot branching, accelerates leaf senescence, and regulates lamina joint angle in rice. Here we show biomass, total S contents, and SL levels in rice under S-sufficient and S-deficient conditions using a split-root system. When one part of the root system was cultured in S-sufficient medium and the other in S-deficient medium (+S/-S), shoot fresh weight was unaffected relative to the +S/+S condition. The shoot weight significantly decreased in -S/-S condition. In contrast, there was no significant difference in root fresh weight between +S and -S conditions. In +S/-S condition, SL levels were systemically reduced in both parts, the shoot S content increased, but the root S content in S-deficient medium was unaffected relative to the -S/-S condition. These results suggest that shoots, not roots, recognize S deficiency, which induces SL production in roots.

Contribution of strigolactones to the inhibition of tiller bud outgrowth under phosphate deficiency in rice.

Strigolactones (SLs) or SL-derived metabolite(s) have recently been shown to act as endogenous inhibitors of axillary bud outgrowth. SLs released from roots induce hyphal branching of arbuscular mycorrhizal (AM) fungi that facilitate the uptake of inorganic nutrients, such as phosphate (Pi) and nitrate, by the host plants. Previous studies have shown that SL levels in root exudates are highly elevated by Pi starvation, which might contribute to successful symbiosis with AM fungi in the rhizosphere. However, how endogenous SL levels elevated by Pi starvation contribute to its hormonal action has been unknown. Here, we show that tiller bud outgrowth in wild-type rice seedlings is inhibited, while root 2'-epi-5-deoxystrigol (epi-5DS) levels are elevated, in response to decreasing Pi concentrations in the media. However, the suppression of tiller bud outgrowth under Pi deficiency does not occur in the SL-deficient and -insensitive mutants. We also show that the responsiveness to exogenous SL is slightly increased by Pi deficiency. When Pi-starved seedlings are transferred to Pi-sufficient media, tiller bud outgrowth is induced following a decrease in root epi-5DS levels. Taken together, these results suggest that elevated SL levels by Pi starvation contribute to the inhibition of tiller bud outgrowth in rice seedlings. We speculate that SL plays a dual role in the adaptation to Pi deficiency; one as a rhizosphere signal to maximize AM fungi symbiosis for improved Pi acquisition and the other as an endogenous hormone or its biosynthetic precursor to optimize shoot branching for efficient Pi utilization.

A new lead chemical for strigolactone biosynthesis inhibitors.

Several triazole-containing chemicals have previously been shown to act as efficient inhibitors of cytochrome P450 monooxygenases. To discover a strigolactone biosynthesis inhibitor, we screened a chemical library of triazole derivatives to find chemicals that induce tiller bud outgrowth of rice seedlings. We discovered a triazole-type chemical, TIS13 [2,2-dimethyl-7-phenoxy-4-(1H-1,2,4-triazol-1-yl)heptan-3-ol], which induced outgrowth of second tiller buds of wild-type seedlings, as observed for non-treated strigolactone-deficient d10 mutant seedlings. TIS13 treatment reduced strigolactone levels in both roots and root exudates in a concentration-dependent manner. Co-application of GR24, a synthetic strigolactone, with TIS13 canceled the TIS13-induced tiller bud outgrowth. Taken together, these results indicate that TIS13 inhibits strigolactone biosynthesis in rice seedlings. We propose that TIS13 is a new lead compound for the development of specific strigolactone biosynthesis inhibitors.

FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice.

Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 microM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 microM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice.

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations.

BACKGROUND AND AIMS: Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections. METHODS: Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population. KEY RESULTS: Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal sigma(g) and neighbourhood size N(b) were estimated to be 4-12 m [95 % confidence interval (CI): 3-25] and 42-73 plants (95 % CI: 28-322) in Meijendel and 10-31 m (95 % CI: 7-infinity) and 57-198 plants (95 % CI: 28-infinity) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %. CONCLUSIONS: SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.

Strigolactones Decrease Leaf Angle in Response to Nutrient Deficiencies in Rice.

Strigolactones (SLs) are a class of plant hormones that are synthesized from ß-carotene through sequential reactions catalyzed by DWARF (D) 27, D17, D10, and OsMORE AXILLARY GROWTH (MAX) 1 in rice (Oryza sativa L.). In rice, endogenous SL levels increase in response to deficiency of nitrogen, phosphate, or sulfate (-N, -P, or -S). Rice SL mutants show increased lamina joint (LJ) angle as well as dwarfism, delayed leaf senescence, and enhanced shoot branching. The LJ angle is an important trait that determines plant architecture. To evaluate the effect of endogenous SLs on LJ angle in rice, we measured LJ angle and analyzed the expression of SL-biosynthesis genes under macronutrient deficiencies. In the "Shiokari" background, LJ angle was significantly larger in SL mutants than in the wild-type (WT). In WT and SL-biosynthesis mutants, direct treatment with the SL synthetic analog GR24 decreased the LJ angle. In WT, deficiency of N, P, or S, but not of K, Ca, Mg, or Fe decreased LJ angle. In SL mutants, deficiency of N, P, or S had no such effect. We analyzed the time course of SL-related gene expression in the LJ of WT deficient in N, P, or S, and found that expression of SL-biosynthesis genes increased 2 or 3 days after the onset of deficiency. Expression levels of both the SL-biosynthesis and signaling genes was particularly strongly increased under -P. Rice cultivars "Nipponbare", "Norin 8", and "Kasalath" had larger LJ angle than "Shiokari", interestingly with no significant differences between WT and SL mutants. In "Nipponbare", endogenous SL levels increased and the LJ angle was decreased under -N and -P. These results indicate that SL levels increased in response to nutrient deficiencies, and that elevated endogenous SLs might negatively regulate leaf angle in rice.

Nanometer accuracy statistical interferometric technique in monitoring the short-term effects of exogenous plant hormones, auxin and gibberellic acid, on rice plants.

Statistical interferometric technique (SIT) is a highly sensitive, high speed non-contact, and non-destructive optical technique developed by our group capable of measuring instantaeoues sub-nanometer displacements. SIT applied to plant leaf elongation revealed nanometric intrinsic fluctuaitons (NIF) that are robust and sensitive to variations in the environment making NIF as a measure of healthiness of the plants. In this study, exogenous plant hormones, auxin (2,4-dichlorophenoxyacetic acid-2,4-D), and gibberellic acid (GA3), along with an auxin transport inhibitor 2,3,5-triiodobenzoic acid-TIBA, that affect plant growth were used to investigate their effects on NIF. Rice (Oriza sativa) seedlings were used, and their roots were exposed to 1, 2, and 4 µM 2,4-D, and the auxin transport inhibitor, TIBA, of 10, and 20 µM for 22 h and GA3 solution of different concentrations of 10, 40, and 100 µM for 5 h. Results showed significant increment in NIF for 1 µM and reduction for 4 µM 2,4-D while applicaiton of both 10, and 20 µM TIBA led to reduction in NIF. On the other hand, significant increment in NIF for 40 µM, and a significant reduction at a higher concentration of 100 µM for 5 hours of GA3 were also observed in comparison to those of control. Our results indicate that NIF as revealed by SIT could show both the positive and negative effects depending on the concentration of exogenous hormones, and transport inhibitors. Results suggest that SIT could be a valuable tool being sensitive enough to speedily assess the effects of plant growth hormones.

Hydroxyl carlactone derivatives are predominant strigolactones in Arabidopsis.

Strigolactones (SLs) regulate important aspects of plant growth and stress responses. Many diverse types of SL occur in plants, but a complete picture of biosynthesis remains unclear. In Arabidopsis thaliana, we have demonstrated that MAX1, a cytochrome P450 monooxygenase, converts carlactone (CL) into carlactonoic acid (CLA) and that LBO, a 2-oxoglutarate-dependent dioxygenase, can convert methyl carlactonoate (MeCLA) into a metabolite called [MeCLA + 16 Da]. In the present study, feeding experiments with deuterated MeCLAs revealed that [MeCLA + 16 Da] is hydroxymethyl carlactonoate (1'-HO-MeCLA). Importantly, this LBO metabolite was detected in plants. Interestingly, other related compounds, methyl 4-hydroxycarlactonoate (4-HO-MeCLA) and methyl 16-hydroxycarlactonoate (16-HO-MeCLA), were also found to accumulate in lbo mutants. 3-HO-, 4-HO-, and 16-HO-CL were detected in plants, but their expected corresponding metabolites, HO-CLAs, were absent in max1 mutants. These results suggest that HO-CL derivatives may be predominant SLs in Arabidopsis, produced through MAX1 and LBO.

d14, a strigolactone-insensitive mutant of rice, shows an accelerated outgrowth of tillers.

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 alpha/beta-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.