BRZ-INSENSITIVE-LONG HYPOCOTYL8 inhibits kinase-mediated phosphorylation to regulate brassinosteroid signaling.

Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved serine/threonine protein kinase in eukaryotes. In plants, the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) functions as a central signaling node through which hormonal and environmental signals are integrated to regulate plant development and stress adaptation. BIN2 plays a major regulatory role in brassinosteroid (BR) signaling and is critical for phosphorylating/inactivating BRASSINAZOLE-RESISTANT 1 (BZR1), also known as BRZ-INSENSITIVE-LONG HYPOCOTYL 1 (BIL1), a master transcription factor of BR signaling, but the detailed regulatory mechanism of BIN2 action has not been fully revealed. In this study, we identified BIL8 as a positive regulator of BR signaling and plant growth in Arabidopsis (Arabidopsis thaliana). Genetic and biochemical analyses showed that BIL8 is downstream of the BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and promotes the dephosphorylation of BIL1/BZR1. BIL8 interacts with and inhibits the activity of the BIN2 kinase, leading to the accumulation of dephosphorylated BIL1/BZR1. BIL8 suppresses the cytoplasmic localization of BIL1/BZR1, which is induced via BIN2-mediated phosphorylation. Our study reveals a regulatory factor, BIL8, that positively regulates BR signaling by inhibiting BIN2 activity.

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation.

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

BIL9 Promotes Both Plant Growth via BR Signaling and Drought Stress Resistance by Binding with the Transcription Factor HDG11.

Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.

Biomimetic Mineralization for Smart Biostimulant Delivery and Crop Micronutrients Fortification.

Sustainable and precise fortification practices are necessary to ensure food security for the increasing human population. Precision agriculture aims to minimize the use of fertilizers and pesticides by developing smart materials for real-life agricultural practices. Here, we show that biomimetic mineralization can be efficiently employed to encapsulate and controllably release plant biostimulants (MiZax-3) to improve the quality and yield of capsicum (Capsicum annum) crops in field experiments. ZIF-8 encapsulation of MiZax-3 (MiZIFs) could significantly enhance its stability up to around 679 times (6p value = 0.0072) at field conditions. Our results demonstrate that the coordinating Zn ions and the MiZax-3 play a vital role in improving Zn content in the produced fruits by 2-fold, which is the first report of this nature on Zn content in fruits. We envision this platform as a starting point to investigate other biocompatible coordination-based platforms for micronutrient delivery in precision agriculture.

Priming of Immune System in Tomato by Treatment with Low Concentration of L-Methionine.

Various metabolites, including phytohormones, phytoalexins, and amino acids, take part in the plant immune system. Herein, we analyzed the effects of L-methionine (Met), a sulfur-containing amino acid, on the plant immune system in tomato. Treatment with low concentrations of Met enhanced the resistance of tomato to a broad range of diseases caused by the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and the necrotrophic fungal pathogen Botrytis cinerea (Bc), although it did not induce the production of any antimicrobial substances against these pathogens in tomato leaf tissues. Analyses of gene expression and phytohormone accumulation indicated that Met treatment alone did not activate the defense signals mediated by salicylic acid, jasmonic acid, and ethylene. However, the salicylic acid-responsive defense gene and the jasmonic acid-responsive gene were induced more rapidly in Met-treated plants after infection with Pst and Bc, respectively. These findings suggest that low concentrations of Met have a priming effect on the phytohormone-mediated immune system in tomato.

Rational design of Striga hermonthica-specific seed germination inhibitors.

The obligate hemiparasitic weed Striga hermonthica grows on cereal roots and presents a severe threat to global food security by causing enormous yield losses, particularly in sub-Saharan Africa. The rapidly increasing Striga seed bank in infested soils provides a major obstacle in controlling this weed. Striga seeds require host-derived strigolactones (SLs) for germination, and corresponding antagonists could be used as germination inhibitors. Recently, we demonstrated that the common detergent Triton X-100 is a specific inhibitor of Striga seed germination by binding noncovalently to its receptor, S. hermonthica HYPO-SENSITIVE TO LIGHT 7 (ShHTL7), without blocking the rice (Oryza sativa) SL receptor DWARF14 (OsD14). Moreover, triazole ureas, the potent covalently binding antagonists of rice SL perception with much higher activity toward OsD14, showed inhibition of Striga but were less specific. Considering that Triton X-100 is not suitable for field application and by combining structural elements of Triton and triazole urea, we developed two hybrid compounds, KK023-N1 and KK023-N2, as potential Striga-specific germination inhibitors. Both compounds blocked the hydrolysis activity of ShHTL7 but did not affect that of OsD14. Binding of KK023-N1 diminished ShHTL7 interaction with S. hermonthica MORE AXILLARY BRANCHING 2, a major component in SL signal transduction, and increased ShHTL7 thermal specificity. Docking studies indicate that KK023-N1 binding is not covalent but is caused by hydrophobic interactions. Finally, in vitro and greenhouse tests revealed specific inhibition of Striga seed germination, which led to a 38% reduction in Striga infestation in pot experiments. These findings reveal that KK023-N1 is a potential candidate for combating Striga and a promising basis for rational design and development of further Striga-specific herbicides.

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.

Analysis of the effect of each plant hormone on the maturation of woodland strawberry fruit in auxin-induced parthenocarpic fruit.

Evaluation of individual roles of plant hormones in fruit development is difficult because various plant hormones function simultaneously. In this study, to analyze the effect of plant hormones on fruit maturation one by one, plant hormones were applied to auxin-induced parthenocarpic woodland strawberry (Fragaria vesca) fruits. As a result, auxin, gibberellin (GA), and jasmonate, but, not abscisic acid and ethylene increased the proportion of ultimately mature fruits. So far, to produce comparable fruit with pollinated fruit in size, auxin with GA treatment was required in woodland strawberry. Picrolam (Pic), the most potent auxin in inducing parthenocarpic fruit, induced fruit which is comparable in size with pollinated fruit without GA. The endogenous GA level and the result of the RNA interference analysis of the main GA biosynthetic gene suggest that a basal level of endogenous GA is essential for fruit development. The effect of other plant hormones was also discussed.

Chemical screening of inhibitors specific for MdDOX-Co that cause an apple columnar tree-shape.

MdDOX-Co, the ectopic expression of which is considered to cause the apple columnar tree shape, belongs to the 2-oxoglutarate-dependent dioxygenase (2ODD) family. It adds a hydroxyl group to position 12 of gibberellins (GAs). However, the 2ODD enzymes related to GA biosynthesis and catabolism are phylogenetically distinct from MdDOX-Co. Thus, it is possible that substrates other than GAs exist in MdDOX-Co. To identify the previously unidentified substrate(s) of MdDOX-Co, we searched for MdDOX-Co-specific inhibitors. Chemical screening using gas chromatography-mass spectrometry was performed to investigate the effects of 2400 compounds that inhibited the catalytic reaction of MdDOX-Co, but not the catabolic reaction of GA 2-oxidase, an enzyme involved in GA catabolism. By applying two positive compounds in Arabidopsis, a chemical 3-((2-chloro-6-fluorobenzyl)thio)-5,7-dimethyl-5H-pyrazolo[3,4-e][1,4,2]dithiazine-1,1-dioxide designated as TPDD that did not inhibit GA biosynthesis was selected. The structure-activity relationships among the TPDD analogs were also obtained.

Small Molecules with Thiourea Skeleton Induce Ethylene Response in Arabidopsis.

Ethylene is the only gaseous plant hormone that regulates several aspects of plant growth, from seedling morphogenesis to fruit ripening and organ senescence. Ethylene also stimulates the germination of Striga hermonthica, a root parasitic weed that severely damages crops in sub-Saharan Africa. Thus, ethylene response stimulants can be used as weed and crop control agents. Ethylene and ethephon, an ethylene-releasing compound, are currently used as ethylene response inducers. However, since ethylene is a gas, which limits its practical application, we targeted the development of a solid ethylene response inducer that could overcome this disadvantage. We performed chemical screening using Arabidopsis thaliana "triple response" as an indicator of ethylene response. After screening, we selected a compound with a thiourea skeleton and named it ZKT1. We then synthesized various derivatives of ZKT1 and evaluated their ethylene-like activities in Arabidopsis. Some derivatives showed considerably higher activity than ZKT1, and their activity was comparable to that of 1-aminocyclopropane-1-carboxylate. Mode of action analysis using chemical inhibitors and ethylene signaling mutants revealed that ZKT1 derivatives activate the ethylene signaling pathway through interactions with its upstream components. These thiourea derivatives can potentially be potent crop-controlling chemicals.

Characterization of Disease Resistance Induced by a Pyrazolecarboxylic Acid Derivative in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid (SA)-mediated signaling pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in Arabidopsis. The soil drench application of CMPA enhanced a broad range of disease resistance against the bacterial pathogen Pseudomonas syringae and fungal pathogens Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis, whereas CMPA did not show antibacterial activity. Foliar spraying with CMPA induced the expression of SA-responsible genes such as PR1, PR2 and PR5. The effects of CMPA on resistance against the bacterial pathogen and the expression of PR genes were observed in the SA biosynthesis mutant, however, while they were not observed in the SA-receptor-deficient npr1 mutant. Thus, these findings indicate that CMPA induces SAR by triggering the downstream signaling of SA biosynthesis in the SA-mediated signaling pathway.

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively; (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 ; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 ; (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 ; (v) no GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.

Function of hydroxycinnamoyl spermidines in seedling growth of Arabidopsis.

Hydroxycinnamic acid amides are involved in various developmental processes as well as in biotic and abiotic stress responses. Among them, the presence of spermidine derivatives, such as N1,N8-di(coumaroyl)-spermidine and N1,N8-di(sinapoyl)-spermidine, and their biosynthetic genes have been reported in Arabidopsis, but their functions in plants are still unknown. We chemically synthesized the above-mentioned spermidine derivatives to assess their physiological functions in Arabidopsis. We evaluated the growth and development of chemically treated Arabidopsis and demonstrated that these compounds inhibited seed germination, hypocotyl elongation, and primary root growth, which could be due to modulation of plant hormone homeostasis and signaling. The results suggest that these compounds are regulatory metabolites that modulate plant growth and development.

BRZ-INSENSITIVE-PALE GREEN 1 is encoded by chlorophyll biosynthesis enzyme gene that functions in the downstream of brassinosteroid signaling.

Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.

Strigolactones Modulate Salicylic Acid-Mediated Disease Resistance in Arabidopsis thaliana.

Strigolactones are low-molecular-weight phytohormones that play several roles in plants, such as regulation of shoot branching and interactions with arbuscular mycorrhizal fungi and parasitic weeds. Recently, strigolactones have been shown to be involved in plant responses to abiotic and biotic stress conditions. Herein, we analyzed the effects of strigolactones on systemic acquired resistance induced through salicylic acid-mediated signaling. We observed that the systemic acquired resistance inducer enhanced disease resistance in strigolactone-signaling and biosynthesis-deficient mutants. However, the amount of endogenous salicylic acid and the expression levels of salicylic acid-responsive genes were lower in strigolactone signaling-deficient max2 mutants than in wildtype plants. In both the wildtype and strigolactone biosynthesis-deficient mutants, the strigolactone analog GR24 enhanced disease resistance, whereas treatment with a strigolactone biosynthesis inhibitor suppressed disease resistance in the wildtype. Before inoculation of wildtype plants with pathogenic bacteria, treatment with GR24 did not induce defense-related genes; however, salicylic acid-responsive defense genes were rapidly induced after pathogenic infection. These findings suggest that strigolactones have a priming effect on Arabidopsis thaliana by inducing salicylic acid-mediated disease resistance.

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.

Induction of tocopherol biosynthesis through heat shock treatment in Arabidopsis.

Plants have developed various self-defense systems to survive many types of unfavorable conditions. Heat shock (HS) treatment, an abiotic stress, activates salicylic acid (SA) biosynthesis to enhance resistance to biotic stresses in some plant species. Since SA is produced from the shikimate pathway, other related metabolic pathways were expected to be upregulated by HS treatment. We speculated that tocopherol biosynthesis utilizing chorismic acid would be activated by HS treatment. In Arabidopsis, expression analysis of tocopherol biosynthetic genes, HPPD, VTE2, VTE3, VTE1, and VTE4, in combination with measurement of metabolites, indicated that HS treatment enhanced the biosynthesis and accumulation of tocopherols. Analyses using an SA biosynthesis-deficient mutant indicated that the upregulation of tocopherol biosynthesis was independent of the SA-mediated signaling pathway.

Light Activates Brassinosteroid Biosynthesis to Promote Hook Opening and Petiole Development in Arabidopsis thaliana.

Although brassinosteroids (BRs) have been proposed to be negative regulators of photomorphogenesis, their physiological role therein has remained elusive. We studied light-induced photomorphogenic development in the presence of the BR biosynthesis inhibitor, brassinazole (Brz). Hook opening was inhibited in the presence of Brz; this inhibition was reversed in the presence of brassinolide (BL). Hook opening was accompanied by cell expansion on the inner (concave) side of the hook. This cell expansion was inhibited in the presence of Brz but was restored upon the addition of BL. We then evaluated light-induced organ-specific expression of three BR biosynthesis genes, DWF4, BR6ox1 and BR6ox2, and a BR-responsive gene, SAUR-AC1, during the photomorphogenesis of Arabidopsis. Expression of these genes was induced, particularly in the hook region, in response to illumination. The induction peaked after 3 h of light exposure and preceded hook opening. Phytochrome-deficient mutants, hy1, hy2 and phyAphyB, and a light-signaling mutant, hy5, were defective in light-induced expression of BR6ox1, BR6ox2 and SAUR-AC1. Light induced both expression of BR6ox genes and petiole development. Petiole development was inhibited in the presence of Brz. Our results largely contradict the early view that BRs are negative regulators of photomorphogenesis. Our data collectively suggest that light activates the expression of BR biosynthesis genes in the hook region via a phytochrome-signaling pathway and HY5 and that BR biosynthesis is essential for hook opening and petiole development during photomorphogenesis.

Triflumizole as a Novel Lead Compound for Strigolactone Biosynthesis Inhibitor.

Strigolactones (SLs) are carotenoid-derived plant hormones involved in the development of various plants. SLs also stimulate seed germination of the root parasitic plants, Striga spp. and Orobanche spp., which reduce crop yield. Therefore, regulating SL biosynthesis may lessen the damage of root parasitic plants. Biosynthetic inhibitors effectively control biological processes by targeted regulation of biologically active compounds. In addition, biosynthetic inhibitors regulate endogenous levels in developmental stage- and tissue-specific manners. To date, although some chemicals have been found as SL biosynthesis inhibitor, these are derived from only three lead chemicals. In this study, to find a novel lead chemical for SL biosynthesis inhibitor, 27 nitrogen-containing heterocyclic derivatives were screened for inhibition of SL biosynthesis. Triflumizole most effectively reduced the levels of rice SL, 4-deoxyorobanchol (4DO), in root exudates. In addition, triflumizole inhibited endogenous 4DO biosynthesis in rice roots by inhibiting the enzymatic activity of Os900, a rice enzyme that converts the SL intermediate carlactone to 4DO. A Striga germination assay revealed that triflumizole-treated rice displayed a reduced level of germination stimulation for Striga. These results identify triflumizole as a novel lead compound for inhibition of SL biosynthesis.

Synthetic agonist of HTL/KAI2 shows potent stimulating activity for Arabidopsis seed germination.

HTL/KAI2, a member of the α/ß-fold hydrolase superfamily, is known to be a receptor-like protein of lactone compounds and that triggers seed germination of Arabidopsis. However, the endogenous ligand and physiological roles of HTL/KAI2 have remained unclear. To understand the mechanism underlying seed germination involved in HTL/KAI2 signaling, it is necessary to identify the endogenous ligand of HTL/KAI2. To date, even a biosynthetic mutant of the ligand has not yet been isolated. Because exogenous agonistic chemicals can only be purchased in small amounts at high prices, the limited supply of those chemicals has hampered any large-scale experiments, such as mutant screening. Therefore, easily synthesized and scalable artificial agonist would remove the limitation of the chemical supply and contribute to the identification of the endogenous ligand of HTL/KAI2 and/or the biosynthetic mutants. In this study, we demonstrated that designed chemicals with a phenoxyfuranone scaffold potently stimulated seed germination via HTL/KAI2 in Arabidopsis. As a result of screening of these chemicals, we selected a representative compound with convincing selectivity. Here in, we provide a new promising synthetic agonist of HTL/KAI2.