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.

Inhibition of NPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions in Arabidopsis.

Under low-Ca conditions, plants accumulate salicylic acid (SA) and induce SA-responsive genes. However, the relationship between SA and low-Ca tolerance remains unclear. Here, we demonstrated that the inhibition or suppression of nonexpressor of pathogenesis-related 1 (NPR1) activity, a major regulator of the SA signaling pathway in the defense response, improves shoot growth under low-Ca conditions. Furthermore, mutations in phytoalexin-deficient 4 (PAD4) or enhanced disease susceptibility 1 (EDS1), which are upstream regulators of NPR1, improved shoot growth under low-Ca conditions, suggesting that NPR1 suppressed growth under low-Ca conditions. In contrast, growth of SA induction-deficient 2-2 (sid2-2), which is an SA-deficient mutant, was sensitive to low Ca levels, suggesting that SA accumulation by SID2 was not related to growth inhibition under low-Ca conditions. Additionally, npr1-1 showed low-Ca tolerance, and the application of tenoxicam-an inhibitor of the NPR1-mediated activation of gene expression-also improved shoot growth under low Ca conditions. The low-Ca tolerance of double mutants pad4-1, npr1-1 and eds1-22 npr1-1 was similar to that of the single mutants, suggesting that PAD4 and EDS1 are involved in the same genetic pathway in suppressing growth under low-Ca conditions as NPR1. Cell death and low-Ca tolerance did not correlate among the mutants, suggesting that growth improvement in the mutants was not due to cell death inhibition. In conclusion, we revealed that NPR1 suppresses plant growth under low-Ca conditions and that the other SA-related genes influence plant growth and cell death.

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.

Emerging technologies for the chemical control of root parasitic weeds.

Parasitic plants in the Orobanchaceae family include devastating weed species, such as Striga, Orobanche, and Phelipanche, which parasitize major crops, drastically reduces crop yields and cause economic losses of over a billion US dollars worldwide. Advances in basic research on molecular and cellular processes responsible for parasitic relationships has now achieved steady progress through advances in genome analysis, biochemical analysis and structural biology. On the basis of these advances it is now possible to develop chemicals that control parasitism and reduce agricultural damage. In this review we summarized the recent development of chemicals that can control each step of parasitism from strigolactone biosynthesis in host plants to haustorium formation.

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.

Germination Stimulant Activity of Isothiocyanates on Phelipanche spp.

The root parasitic weed broomrapes, Phelipanche spp., cause severe damage to agriculture all over the world. They have a special host-dependent lifecycle and their seeds can germinate only when they receive chemical signals released from host roots. Our previous study demonstrated that 2-phenylethyl isothiocyanate is an active germination stimulant for P. ramosa in root exudates of oilseed rape. In the present study, 21 commercially available ITCs were examined for P. ramosa seed germination stimulation, and some important structural features of ITCs for exhibiting P. ramosa seed germination stimulation have been uncovered. Structural optimization of ITC for germination stimulation resulted in ITCs that are highly active to P. ramosa. Interestingly, these ITCs induced germination of P. aegyptiaca but not Orobanche minor or Striga hermonthica. P. aegyptiaca seeds collected from mature plants parasitizing different hosts responded to these ITCs with different levels of sensitivity. ITCs have the potential to be used as inducers of suicidal germination of Phelipanche seeds.

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.

Chemical Synthesis of Triazole-Derived Suppressors of Strigolactone Functions.

Triazole is a five-membered heteroring consists of two carbon atoms and three nitrogen atoms and exhibits a wide range of biological activities. The basic heterocyclic rings are 1,2,3-triazole and 1,2,4-triazole. Here we describe the chemical synthetic methods for triazole derivatives that can suppress the function of SL by inhibiting SL biosynthesis pathway or SL perception sites such as D14.

Triazole Ureas Covalently Bind to Strigolactone Receptor and Antagonize Strigolactone Responses.

Strigolactones, a class of plant hormones with multiple functions, mediate plant-plant and plant-microorganism communications in the rhizosphere. In this study, we developed potent strigolactone antagonists, which covalently bind to the strigolactone receptor D14, by preparing an array of triazole urea compounds. Using yeast two-hybrid and rice-tillering assays, we identified a triazole urea compound KK094 as a potent inhibitor of strigolactone receptors. Liquid chromatography-tandem mass spectrometry analysis and X-ray crystallography revealed that KK094 was hydrolyzed by D14, and that a reaction product of this degradation covalently binds to the Ser residue of the catalytic triad of D14. Furthermore, we identified two triazole urea compounds KK052 and KK073, whose effects on D14-D53/D14-SLR1 complex formation were opposite due to the absence (KK052) or presence (KK073) of a trifluoromethyl group on their phenyl ring. These results demonstrate that triazole urea compounds are potentially powerful tools for agricultural application and may be useful for the elucidation of the complicated mechanism underlying strigolactone perception.

Structural analysis of HTL and D14 proteins reveals the basis for ligand selectivity in Striga.

HYPOSENSITIVE TO LIGHT (HTL) and DWARF14 (D14) mediate the perception of karrikin and strigolactone, which stimulates germination of the parasitic weed Striga. However, their role in parasitic seeds is poorly understood, and the basis for their differing responsiveness remains unclear. Here, we show that Striga hermonthica HTL proteins (ShHTLs) in 'conserved' and 'intermediate' clades are able to bind karrikin. The 'divergent' clade is able to hydrolyze strigolactone. Unexpectedly, we find that ShD14 is also capable of hydrolyzing strigolactone. Through comparative analysis of ShHTLs and ShD14 crystal structures, we provide insights into the basis for their selectivity. Moreover, we show that both ShD14 and divergent clade ShHTLs, but not conserved and intermediate clade ShHTLs, can interact with the putative downstream signaling component ShMAX2 in the presence of the synthetic strigolactone, rac-GR24. These findings provide insight into how strigolactone is perceived and how ligand specificity is determined.