Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

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.

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.

Additive effects of touch-activated polymerization and extended irradiation time on bonding of light-activated adhesives to root canal dentin.

STATEMENT OF PROBLEM: The bonding of light-activated adhesives to root canal dentin with an additional touch-polymerization activator has been insufficiently examined. PURPOSE: The purpose of this in vitro study was to investigate the effect of touch-polymerization activators and extended light-irradiation time on the microtensile bond strength (µTBS) of light-activated adhesives. MATERIAL AND METHODS: Post cavities were prepared in 50 extracted mandibular premolars and bonded using Prime&Bond Universal (PBU); PBU+Self Cure Activator (SCA); Clearfil SE Bond 2 (SEB); SEB+Clearfil DC Activator (DCA); or Clearfil Universal Bond Quick ER (UBQ). After light-irradiation for 10 or 20 seconds, the post cavities were filled with dual-activated resin core materials. Eight beams were prepared per specimen and subjected to the µTBS test. The µTBS data were analyzed by using 3-way ANOVAs with the Bonferroni correction (α=.05). RESULTS: The 3-way ANOVAs indicated that the use of touch-polymerization activators (SCA and DCA) significantly increased the µTBS of PBU and SEB in both the coronal (P=.015) and apical (P=.001) regions. The extension of light-irradiation time to 20 seconds significantly improved their µTBS in the apical region (P<.001), but not in the coronal region (P=.09). Light-irradiation for 20 seconds increased the µTBS of UBQ significantly in the coronal region (P=.014). CONCLUSIONS: Touch-polymerization activators improved the bond strength of light-activated adhesives to root canal dentin, especially when combined with an extended light-irradiation time.

Replacing mandibular central incisors with a direct resin-bonded fixed dental prosthesis by using a bilayering composite resin injection technique with a digital workflow: A dental technique.

A straightforward technique is presented for an interim or short-term definitive esthetic replacement of missing anterior teeth requiring no tooth preparation. Composite resins are injected into transparent silicone indices fabricated from 3-dimensional-printed casts of a digital waxing. The dentin core is formed of a durable short fiber-reinforced injectable composite resin and veneered with an enamel-shade composite resin for enhanced esthetics. Besides being noninvasive, this technique is more straightforward than traditional options, reducing chair time while providing an accurate outcome.

Hormonal Diterpenoids Distinct to Gibberellins Regulate Protonema Differentiation in the Moss Physcomitrium patens.

Plants synthesize gibberellin (GA), a diterpenoid hormone, via ent-kaurenoic acid (KA) oxidation. GA has not been detected in the moss Physcomitrium patens despite its ability to synthesize KA. It was recently shown that a KA metabolite, 3OH-KA, was identified as an active regulator of protonema differentiation in P. patens. An inactive KA metabolite, 2OH-KA, was also identified in the moss, as was KA2ox, which is responsible for converting KA to 2OH-KA. In this review, we mainly discuss the GA biosynthetic gene homologs identified and characterized in bryophytes. We show the similarities and differences between the OH-KA control of moss and GA control of flowering plants. We also discuss using recent genomic studies; mosses do not contain KAO, even though other bryophytes do. This absence of KAO in mosses corresponds to the presence of KA2ox, which is absent in other vascular plants. Thus, given that 2OH-KA and 3OH-KA were isolated from ferns and flowering plants, respectively, vascular plants may have evolved from ancestral bryophytes that originally produced 3OH-KA and GA.

Effect of smear layer deproteinization with HOCl solution on the dentin bonding of conventional and resin-modified glass-ionomer cements.

The effect of smear layer-deproteinizing pretreatment using hypochlorous acid (HOCl) on the micro-shear bond strengths (µSBS) of conventional and resin-modified glass-ionomer cements (GIC) to dentin was investigated and compared with demineralizing pretreatment with polyacrylic acid (PAA). Three GICs: Fuji IX GP Extra (restorative conventional GIC), GC Fuji II LC EM (restorative resin-modified GIC), and GC Fuji Luting EX (luting resin-modified GIC), were used. One hundred fifty human molars were divided into groups (n = 10) according to the cements and dentin pretreatments; no pretreatment (control), 10 s PAA pretreatment, and HOCl pretreatment for 5, 15, or 30 s. After 24 h, µSBS was tested and the data were statistically analyzed using a two-way ANOVA, followed by Tukey's post-hoc test. HOCl pretreatment significantly increased µSBS of conventional GIC compared to the control group. For resin-modified restorative GIC, 5 s HOCl deproteinization significantly increased µSBS, while longer application times did not. There was no significant difference between HOCl-pretreated and control groups of resin-modified luting GIC. PAA pretreatment increased the µSBS of all cements significantly. In conclusion, smear layer deproteinization with HOCl can enhance the dentin bonding of conventional GIC. However, the residual radicals may adversely affect the polymerization of resin-modified GICs.

The combined effect of light-illuminating direction and enamel rod orientation on color adjustment at the enamel borders of composite restorations.

OBJECTIVES: To investigate the effect of light-illuminating direction (from composite or enamel side) on color adjustment at the coronal and cervical enamel borders in composite restorations. MATERIALS AND METHODS: Forty cylindrical holes (3.0-mm diameters) were prepared in bovine enamel disks (1.0-mm thickness). After application of a one-step self-etch adhesive, one of four resin composites (Estelite Asteria, EA; Estelite Pro, EP; Kalore, KA; Clearfil Majesty ES-2 Premium, MJ) was restored in the holes. After 24-h storage, the colors (L*, C*, or h* values) at the restored enamel disks over a black background were measured in a black box using a CIE XYZ camera, spotted with D65 standard illuminant either from coronal or cervical side at 45°/0° geometry. The color shifting rate was calculated at the coronal and cervical enamel borders of the composite restorations, and analyzed by three-way ANOVA with Dunnett's T3 and t test for post hoc analysis (p < 0.05). RESULTS: The light-illuminating directions significantly affected the L* shifting rate at the cervical enamel border in EP and MJ (p < 0.05), and the C* shifting rate at the coronal enamel border in EA, EP, and MJ (p < 0.05). CONCLUSIONS: The color appearance at the border of the composite restoration was influenced by the light-illuminating direction in conjunction with the enamel rod orientation in the coronal or cervical enamel border. CLINICAL RELEVANCE: The line-of-vision angle would affect the perception of color adaptation at the enamel borders in the composite restorations.

Post-orthodontic recontouring of anterior teeth using composite injection technique with a digital workflow.

OBJECTIVE: Restorative treatment of anterior teeth is often required as the final step of orthodontic therapy to optimize the esthetics and function. This case report presents a direct minimally invasive approach for post-orthodontic recontouring of anterior teeth using the composite injection technique with a digital workflow. CLINICAL CONSIDERATIONS: The extraction of central incisors with short roots was indicated to resolve maxillary anterior crowding. The gained space was closed with lateral incisors, and it was necessary to recontour them and canines to resemble central and lateral incisors, respectively. The restorations were digitally designed, and a transparent silicone index was fabricated from a 3D-printed cast of the digital wax-up. After lateral incisors and canines were bonded with a universal adhesive in the etch-and-rinse mode, a highly filled injectable composite resin was applied into the index. The restorative treatment was preceded by gingivectomy using an Er:YAG laser and home bleaching. CONCLUSIONS: In cases where more complex recontouring of anterior teeth is necessary, the composite injection technique could be a suitable alternative to indirect restorations, because it is straightforward, cost-effective, and does not require any preparation. The digital workflow simplified and expedited the treatment while contributing to its precision. CLINICAL SIGNIFICANCE: Post-orthodontic recontouring using direct composite restorations is well accepted by patients due to low cost and non-invasiveness. However, in complicated cases, the free-hand technique is time-consuming and technique-sensitive. The presented composite injection technique with a digital workflow significantly simplifies and expedites the composite placement while predictably enhancing the treatment outcome.

Substituted Phthalimide AC94377 Is a Selective Agonist of the Gibberellin Receptor GID1.

Gibberellin (GA) is a major plant hormone that regulates plant growth and development and is widely used as a plant growth regulator in agricultural production. There is an increasing demand for function-limited GA mimics due to the limitations on the agronomical application of GA to crops, including GA's high cost of producing and its leading to the crops' lodging. AC94377, a substituted phthalimide, is a chemical that mimics the growth-regulating activity of GAs in various plants, despite its structural difference. Although AC94377 is widely studied in many weeds and crops, its mode of action as a GA mimic is largely unknown. In this study, we confirmed that AC94377 displays GA-like activities in Arabidopsis (Arabidopsis thaliana) and demonstrated that AC94377 binds to the Arabidopsis GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor (AtGID1), forms the AtGID1-AC94377-DELLA complex, and induces the degradation of DELLA protein. Our results also indicated that AC94377 is selective for a specific subtype among three AtGID1s and that the selectivity of AC94377 is attributable to a single residue at the entrance to the hydrophobic pocket of GID1. We conclude that AC94377 is a GID1 agonist with selectivity for a specific subtype of GID1, which could be further developed and used as a function-limited regulator of plant growth in both basic study and agriculture.

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor.

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.

The chemical NJ15 affects hypocotyl elongation and shoot gravitropism via cutin polymerization.

We previously found a chemical, designated as NJ15, which inhibited both auxin and brassinosteroid responses in dark-grown Arabidopsis. To study its mode of action, we performed a phenotypic screening of NJ15-low-sensitive lines among mutant pools of Arabidopsis. One line (f127) showed clear NJ15-low-sensitivity in terms of hypocotyl elongation and shoot gravitropism. After further testing, it was determined that DCR, an enzyme involved in cutin polymerization, had lost its function in the mutant, which caused its low sensitivity to NJ15. Fatty acids are the base materials for polymers such as cutin and cuticular wax. We confirmed that NJ15 affects fatty acid biosynthesis, and that it does differently from cafenstrole, a known inhibitor of cuticular wax formation. Based on these results, we propose that the target of NJ15 is likely located within the cutin polymer formation pathway. ABBREVIATIONS: Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid.

Effect of Polymerization Accelerator on Bond Strength to Eugenol-Contaminated Dentin.

PURPOSE: To evaluate the effect of a polymerization accelerator on the microtensile bond strength (µTBS) of etch-and-rinse and self-etch adhesives to eugenol-contaminated dentin. MATERIALS AND METHODS: Sixty flat dentin surfaces were prepared from human molars. Half of the specimens were restored with zinc oxide eugenol temporary cement (IRM) (eugenol-contaminated group) and the other half remained without restoration (control group). After 24-h storage, the cement was mechanically removed. Then the specimens in each group were further divided into three subgroups based on the application procedure of a polymerization accelerator (p-toluenesulfinic acid sodium salt; Accel): no application, 10-s application, or 30-s application. After air drying, the dentin surfaces were bonded with either a three-step etch-and-rinse adhesive (OptiBond FL) or a two-step self-etch adhesive (Clearfil SE Bond) and restored with composite. After 24-h water storage, the bonded specimens were subjected to the µTBS test. Data were analyzed by three-way ANOVA and Dunnett's T3 test (p < 0.05). RESULTS: The eugenol-contaminated groups had significantly lower µTBS than the control groups with both types of adhesives (p < 0.05), and the application of Accel significantly increased the compromised µTBS to eugenol-contaminated dentin. Optibond FL presented significantly higher µTBS to eugenol-contaminated dentin than did Clearfil SE Bond (p < 0.05). CONCLUSION: The application of a polymerization accelerator on eugenol-contaminated dentin prior to adhesive resin application increased the µTBS of both the three-step etch-and-rinse and two-step self-etch adhesive.

Effect of Water Aging of Adherend Composite on Repair Bond Strength of Nanofilled Composites.

PURPOSE: To evaluate the effect of water aging of adherend composite on repair bond strength to nanofilled composites with specific fillers using different bonding agents. MATERIALS AND METHODS: Three nanofilled composites - Beautifil II with S-PRG filler (BE) / Filtek Supreme ultra with nanocluster filler (SP) / Estelite Σ Quick (ES) - and one microhybrid composite, Clearfil APX (AP), were used in this study. The composite disks were immersed in water for different durations (immediate, 1 week, 2 weeks or 1 month), and then the polished surfaces were treated with one of three bonding agents - no treatment (control), application of Clearfil SE One (SE), application of Clearfil SE One plus Clearfil Porcelain Bond Activator (PB) - then filled with a repair composite. The bonded composite disks were subjected to the microshear bond strength (µSBS) test. Additionally, water sorption (Wsp) and solubility (Wsl) of the resin composite were measured. The µSBS data were was statistically analyzed using a three-way ANOVA and t-test with Bonferroni correction for multiple comparisons. RESULTS: Water aging of adherend composite affected the repair bond strength (p < 0.05). For BE, SP, and ES, application of an adhesive agent improved repair bond strengths to water-aged composites (p < 0.05), but adding a silane coupling agent could not (p > 0.05). For AP, the µSBS significantly increased, with control group < SE group < PB group (p < 0.05). CONCLUSION: Microhybrid composite was a more suitable material for composite repair than nanofilled composite, due to adhesion to exposed, larger silica fillers. S-PRG filler and nanocluster filler in the nanofilled composites played a slight role in improving their repair bonding performances with the bonding agents tested.

Chemical screening and development of novel gibberellin mimics.

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure-activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA.

Helminthosporic acid functions as an agonist for gibberellin receptor.

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure-activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

Bonding Durability of a Self-etch Adhesive to Normal Versus Smear-layer Deproteinized Dentin: Effect of a Reducing Agent and Plant-extract Antioxidant.

PURPOSE: To evaluate the effect of a reducing agent and plant-extract antioxidant on the bonding durability of a self-etch adhesive to normal and NaOCl-treated, smear-layer-deproteinized dentin. MATERIALS AND METHODS: Flat smear-layer-covered dentin surfaces from 60 extracted human molars were prepared by removing the occlusal enamel. The teeth were divided into two groups with or without NaOCl-deproteinizing treatment for 30 s, and further divided into three subgroups as follows: no application of antioxidant, application of Accel (p-toluenesulfinic acid sodium salt solution) for 5 s, or application of rosmarinic acid solution for 5 s. All treated dentin surfaces were bonded with a two-step self-etch adhesive (Clearfil SE Bond) and restored with composite (Clearfil AP-X). The bonded teeth were sectioned into a hourglass-shaped sticks with a composite-dentin bonded interface area of 1.0 mm2. After storage in artificial saliva for 24 h or 1 year, the specimens were subjected to the microtensile bond strength test (n = 15). Data were statistically analyzed with three-way ANOVA, Tukey's post-hoc test, and the t-test (p < 0.05). RESULTS: Without an antioxidant, 1-year storage significantly reduced the bond strengths of the self-etch adhesive to normal and smear-layer-deproteinized dentin compared with those after 24-h storage (p < 0.05). Application of Accel and rosmarinic acid restored the compromised initial bond strengths to smear-layer-deproteinized dentin (p < 0.05), and prevented long-term deterioration of bond strengths to both normal and smear-layer-deproteinized dentin (p > 0.05). CONCLUSION: Application of Accel and rosmarinic acid improved bonding durability of the self-etch adhesive to both normal and smear-layer-deproteinized dentin.

Dentin Bonding Durability of Two-step Self-etch Adhesives with Improved of Degree of Conversion of Adhesive Resins.

PURPOSE: To evaluate (1) the initial and long-term microtensile bond strengths of two-step self-etch adhesives with different degrees of conversion (DC); (2) the elastic modulus of the respective adhesive resins; (3) the water sorption of the respective adhesive resins. MATERIALS AND METHODS: Two two-step self-etch adhesives, Clearfil SE Bond (CSE) and Clearfil SE Bond 2 (CSE2) were used in this study. The DC was determined using ATR/FT-IR with a time-based spectrum analysis. Midcoronal flat dentin surfaces of 24 human molars were prepared with 600-grit SiC paper for microtensile bond strength (µTBS) testing. CSE and CSE2 were applied to the dentin surfaces according to the manufacturer's instructions, followed by composite buildups. The µTBS was measured after water storage for 24 h, 6 months, and 1 year. The elastic modulus (before and after 1 month of water immersion) was determined by the three-point flexural bending test and water sorption values by the water sorption test. RESULTS: CSE2 showed significantly higher DC than CSE. The µTBS of CSE2 was significantly higher than that of CSE in all water storage periods. One-year water storage decreased the µTBS of CSE; however, it did not decrease that of CSE2. Regarding the polymerized adhesive resins, the elastic modulus of CSE2 was significantly higher than that of CSE before and after water immersion (p < 0.001), and the water sorption of CSE was higher than that of CSE2. CONCLUSIONS: The higher DC of adhesive resins of two-step self-etch adhesives resists water aging and improves the initial bond strengths and durability of the resin-dentin bond.