Healing Predictors of Conservative Treatment for Juvenile Osteochondritis Dissecans of the Talus.

OBJECTIVE: To investigate the healing response of juvenile osteochondritis dissecans (JOCD) of the talus after conservative treatment, identify healing predictors, and develop a predictive model for healing. DESIGN: Retrospective study. SETTING: Clinics at a tertiary-level pediatric medical center. PATIENTS: Fifty-five patients (55 ankles) who presented with JOCD. INTERVENTIONS: Patients were managed with cast immobilization followed by activity restriction. MAIN OUTCOME MEASURES: The primary outcome measure of progressive lesion reossification was determined from the latest radiograph, after at least 6 months of nonoperative treatment. Final clinical evaluation was performed by a questionnaire and complementary telephone interview. Multivariate logistic regression was used to determine the influence of age, sex, lesion size, classification, location, duration of symptoms, containment lesion, and the occurrence of cyst-like lesions on healing potential. RESULTS: After nonoperative treatment, 18 (33%) of 55 lesions had failed to progress toward healing. An older age (P = 0.034) and a completely detached but undisplaced (grade III) lesion (P < 0.001) at the time of diagnosis were predictive for the failure of conservative treatment. A multivariate logistic regression best predictor model that included age and grade resulted in the best predicted healing and yielded an area under the curve of 0.920 (P < 0.001). CONCLUSION: In two-thirds of skeletally immature patients, conservative treatment resulted in the progressive healing of JOCD of the talus. For older patients with grade III lesions showing a lower healing probability, surgical treatment should be considered.

Comparative Assessment of Biological Activities of Mistletoes for Cosmetic Applications: Viscum Album Var. Coloratum (Kom.) Ohwi and Loranthus Tanakae Franch. & Sav.

Mistletoes, hemiparasites, contain many components with various biological activities and have been used in cosmetics industry. Loranthacease (1,000 species) and Viscaceae (550 species) have the most dominant species in mistletoes (nearly 1,600 species). It can be expected that the biological activities vary from species to species; therefore, we have tested Viscum album var. coloratum (Kom.) Ohwi (belonging to Santalaceae) and Loranthus tanakae Franch. & Sav. (belonging to Loranthacease) for a comparative study of their cosmetic properties, including antioxidant, antimelanogenic, and antiwrinkle activities. As results, the ethanol extract of L. tanakae had higher phenolic content and showed effective antioxidant activity and elastase inhibition. Meanwhile, the ethanol extract of V. album more effectively inhibited tyrosinase. Comparing with ethanol extracts, the water extracts of both mistletoes showed lower biological efficacy than the ethanol extracts or no significant effect. Thus, these results show that different extracts of mistletoe have different levels of biological activities, presumably because of the differences in their phytochemical profiles and because of the different extraction methods used.

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II.

RATIONALE: Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells, apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 (Ras-interacting protein 1) is required for apical junction clearance, as well as for regulation of Rho GTPase (enzyme that hydrolyzes GTP) activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. OBJECTIVE: The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. METHODS AND RESULTS: Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of endothelial cell apical junctions during vascular tubulogenesis depends on Rasip1, as well as the GTPase Cdc42 (cell division control protein 42 homolog) and the kinase Pak4 (serine/threonine-protein kinase 4). Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all blocks endothelial cell tubulogenesis. By contrast, inactivation of RhoA (Ras homologue gene family member A) signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII (nonmuscle myosin II) either before, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. CONCLUSIONS: Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth.

Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular 'tubulogenesis' are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1(-/-) ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC-EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.

Lactobacillus gasseri BNR17 Ameliorates Dexamethasone-Induced Muscle Loss in BALB/c Mice and C2C12 Myotubes.

This study aimed to investigate the effects and mechanism of Lactobacillus gasseri BNR17, a probiotic strain isolated from human breast milk, on dexamethasone-induced muscle loss in mice and cultured myotubes. BALB/c mice were intraperitoneally injected with dexamethasone, and orally administered L. gasseri BNR17 for 21 days. L. gasseri BNR17 treatment ameliorated dexamethasone-induced decline in muscle function, as evidenced by an increase in forelimb grip strength, treadmill running time, and rotarod retention time in both female and male mice. In addition, L. gasseri BNR17 treatment significantly increased the mass of the gastrocnemius and quadriceps muscles. Dual-energy X-ray absorptiometry showed a significant increase in lean body mass and a decrease in fat mass in both whole body and hind limb after treatment with L. gasseri BNR17. It was found that L. gasseri BNR17 treatment downregulated serum myostatin level and the protein degradation pathway composed of muscle-specific ubiquitin E3 ligases, MuRF1 and MAFbx, and their transcription factor FoxO3. In contrast, L. gasseri BNR17 treatment upregulated serum insulin-like growth factor-1 level and Akt-mTOR-p70S6K signaling pathway involved in protein synthesis in muscle. As a result, L. gasseri BNR17 treatment significantly increased the levels of major muscular proteins such as myosin heavy chain and myoblast determination protein 1. Consistent with in vivo results, L. gasseri BNR17 culture supernatant significantly ameliorated dexamethasone-induced C2C12 myotube atrophy in vitro. In conclusion, L. gasseri BNR17 ameliorates muscle loss by downregulating the protein degradation pathway and upregulating the protein synthesis pathway.

Stepwise arteriovenous fate acquisition during mammalian vasculogenesis.

Arteriovenous (AV) differentiation is a critical step during blood vessel formation and stabilization. Defects in arterial or venous fate lead to inappropriate fusion of vessels, resulting in damaging arteriovenous shunts. While many studies have unraveled the molecular underpinnings that drive AV fate, surprisingly, the spatiotemporal emergence of arteries and veins in mammalian embryos remains unknown. Here, we examine artery and vein specification and differentiation during vasculogenesis. We show that the first intraembryonic vessels formed are arteries, which differentiate in a stepwise manner. By contrast, veins emerge later, progressively forming after embryonic turning. In addition, we demonstrate that hemodynamic flow is not required for arterial specification, but is required for maintenance of select arterial markers. Together, our results provide a first spatiotemporal analysis of mammalian AV cell fate establishment and anatomy, as well as a delineation of a molecular toolkit for analysis of arteries and veins during early vessel development.

Evaluation of the reliability of lower extremity alignment measurements using EOS imaging system while standing in an even weight-bearing posture.

This study aimed to analyze the reproducibility and reliability of the alignment parameters measured using the EOS image system in both limbs while standing with an even weight-bearing posture. Overall, 104 lower extremities in 52 patients were analyzed retrospectively. The patients stood with an even load over both lower extremities then rotated 15° in both directions. Two EOS images were acquired and 104 pairs of lower extremities were compared according to the position of the indexed lower extremities. Then, the inter-observer reliability of the EOS system and the inter-modality reliability between EOS and computed tomography (CT) were evaluated. Femoro-tibial rotation (FTR) and tibial torsion demonstrated a significant difference between the anterior and posterior positions of the indexed lower extremity. In the inter-observer reliability analysis, all values except for FTR and tibial torsion demonstrated good or very good reliability. In the anterior position, FTR demonstrated moderate, and tibial torsion demonstrated poor reliability. In the posterior position, both FTR and tibial torsion demonstrated poor reliability. In the reliability analysis between the three-dimensional (3D) EOS model and 3D CT images, all measurements of the femur demonstrated very good reliability, but measurements of the tibia did not. For the coronal and sagittal alignment parameters measured by the EOS 3D system with rotated standing posture, except for the measurement including tibial torsion., there were no significant difference for either position of the indexed extremities with high agreement between the observers as well as with the CT 3D model.

The effect of hamstring tightness on intraoperative extension gap in posterior stabilized total knee arthroplasty.

This study aimed to determine the factors related to intraoperative extension gap (EG) in patients who underwent posterior-stabilized total knee arthroplasty (TKA). A total of 106 TKAs in 84 patients were retrospectively reviewed. Only patients who underwent the same method of bone resection were included consecutively. Bilateral popliteal angle (BPA) was used as an indicator of hamstring tightness. EG and extension space angle were measured using an offset type tensor. The associations between patient variables and EG were analyzed using multivariable linear regression and Pearson's correlation coefficients. The average EG was 12.9 ± 2.1 mm, and the average extension space angle was 2.8° ± 3.2°. BPA was greater than flexion contracture in most cases (94.3%), and no difference was found in only six cases (5.7%). According to multivariable linear regression analysis which was conducted after modifying the BPA into a categorical variable by 5°, EG was correlated with BPA (p < 0.001). Pearson's correlation coefficient between EG and BPA was - 0.674 (p < 0.001). No other factors were significantly correlated with intraoperative EG. The present study found that popliteal angle is a different entity from flexion contracture, and that it is a predictable factor for EG in osteoarthritis patients. Smaller BPAs led to larger EG in patients who underwent the same degree of bone resection.

Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae.

We reported previously that a recombinant salicylic acid (SA) glucosyltransferase1 (AtSGT1) from Arabidopsis thaliana catalyzes the formation of both SA 2-O-beta-D-glucoside (SAG) and the glucose ester of SA (SGE). Here, transgenic Arabidopsis plants overexpressing AtSGT1 have been constructed, and their phenotypes analyzed. Compared to wild-type plants, transgenic plants showed an increased susceptibility to Pseudomonas syringae and reduced the accumulation levels of both free SA and its glucosylated forms (SAG and SGE). On the other hand, the overexpression increased the levels of methyl salicylate (MeSA) and methyl salicylate 2-O-beta-D-glucoside (MeSAG), and also induced SA carboxyl methyltransferase1 (AtBSMT1) expression, whose products catalyze the conversion of SA to MeSA. Our data indicate that reduced resistance by AtSGT1 overexpression results from a reduction in SA content, which is at least in part caused by increases in MeSAG and MeSA levels at the expense of SA. Our study also suggests that genetic manipulation of AtSGT1 can be utilized as an important regulatory tool for pathogen control.

An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography-chemical ionization-mass spectrometry.

We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.

Effect of Course Structure on the Accuracy of Nonsterile Compounded Preparations.

Objective. To investigate if students in the new course structure attained the same level of compounding competency as students in the legacy course structure. Methods. Students compounded four nonsterile preparations common to both the legacy curriculum (PCL) and the transformed curriculum (TC). The preparations were compared using relative potency or weight variation as a measure of compounding competency. They represented the broad range of compounding complexities required in compounding courses at the school. Results. The mean relative potencies of three nonsterile preparations were statistically different, with only the mean of the TC hydrocortisone medication stick being outside of the acceptable range of the laboratory's criteria. However, the standard deviation (SD) was markedly different in each preparation pair suggesting that the number of students correctly compounding the preparation in the first attempt might be an important factor in the analysis. In contrast, the mean weight variation data of the phenol-menthol soft troches and enalapril tablet triturates were almost identical. Conclusion. The relative potency results suggested that equivalent competency in the two student groups was possible for preparations that involved simple solutions or filled fixed volume molds. However, the hydrocortisone medication stick data indicated that understanding the science of a preparation may require more knowledge or time.

Grain width 2 (GW2) and its interacting proteins regulate seed development in rice (Oryza sativa L.).

BACKGROUND: Seed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size. RESULTS: Proteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical ß-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds. CONCLUSIONS: Collectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling.

Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COMMD-CCDC22-CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COMMD) family members that can associate with the CCC complex, only COMMD9 and its binding partner, COMMD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COMMD protein family.

Autophagy is essential for cardiac morphogenesis during vertebrate development.

Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.

Identification of a novel jasmonate-responsive element in the AtJMT promoter and its binding protein for AtJMT repression.

Jasmonates (JAs) are important regulators of plant biotic and abiotic stress responses and development. AtJMT in Arabidopsis thaliana and BcNTR1 in Brassica campestris encode jasmonic acid carboxyl methyltransferases, which catalyze methyl jasmonate (MeJA) biosynthesis and are involved in JA signaling. Their expression is induced by MeJA application. To understand its regulatory mechanism, here we define a novel JA-responsive cis-element (JARE), G(C)TCCTGA, in the AtJMT and BcNTR1 promoters, by promoter deletion analysis and Yeast 1-Hybrid (Y1H) assays; the JARE is distinct from other JA-responsive cis-elements previously reported. We also used Y1H screening to identify a trans-acting factor, AtBBD1, which binds to the JARE and interacts with AtJAZ1 and AtJAZ4. Knockout and overexpression analyses showed that AtBBD1 and its close homologue AtBBD2 are functionally redundant and act as negative regulators of AtJMT expression. However, AtBBD1 positively regulated the JA-responsive expression of JR2. Chromatin immunoprecipitation from knockout and overexpression plants revealed that repression of AtJMT is associated with reduced histone acetylation in the promoter region containing the JARE. These results show that AtBBD1 interacts with JAZ proteins, binds to the JARE and represses AtJMT expression.

The expression patterns of AtBSMT1 and AtSAGT1 encoding a salicylic acid (SA) methyltransferase and a SA glucosyltransferase, respectively, in Arabidopsis plants with altered defense responses.

We reported previously that overexpression of a salicylic acid (SA) methyltransferase1 gene from rice (OsBSMT1) or a SA glucosyltransferase1 gene from Arabidopsis thaliana (AtSAGT1) leads to increased susceptibility to Pseudomonas syringae due to reduced SA levels. To further examine their roles in the defense responses, we assayed the transcript levels of AtBSMT1 or AtSAGT1 in plants with altered levels of SA and/or other defense components. These data showed that AtSAGT1 expression is regulated partially by SA, or non-expressor of pathogenesis related protein1, whereas AtBSMT1 expression was induced in SA-deficient mutant plants. In addition, we produced the transgenic Arabidopsis plants with RNAi-mediated inhibition of AtSAGT1 and isolated a null mutant of AtBSMT1 and then analyzed their phenotypes. A T-DNA insertion mutation in the AtBSMT1 resulted in reduced methyl salicylate (MeSA) levels upon P. syringae infection. However, accumulation of SA and glucosyl SA was similar in both the atbsmt1 and wild-type plants, indicating the presence of another SA methyltransferase or an alternative pathway for MeSA production. The AtSAGT1-RNAi line exhibited no altered phenotypes upon pathogen infection, compared to wild-type plants, suggesting that (an)other SA glucosyltransferase(s) in Arabidopsis plants may be important for the pathogenesis of P. syringae.

Methyl jasmonate reduces grain yield by mediating stress signals to alter spikelet development in rice.

Jasmonic acid (JA) is involved in plant development and the defense response. Transgenic overexpression of the Arabidopsis (Arabidopsis thaliana) jasmonic acid carboxyl methyltransferase gene (AtJMT) linked to the Ubi1 promoter increased levels of methyl jasmonate (MeJA) by 6-fold in young panicles. Grain yield was greatly reduced in Ubi1:AtJMT plants due to a lower numbers of spikelets and lower filling rates than were observed for nontransgenic (NT) controls. Ubi1:AtJMT plants had altered numbers of spikelet organs, including the lemma/palea, lodicule, anther, and pistil. The loss of grain yield and alteration in spikelet organ numbers were reproduced by treating NT plants with exogenous MeJA, indicating that increased levels of MeJA in Ubi1:AtJMT panicles inhibited spikelet development. Interestingly, MeJA levels were increased by 19-fold in young NT panicles upon exposure to drought conditions, resulting in a loss of grain yield that was similar to that observed in Ubi1:AtJMT plants. Levels of abscisic acid (ABA) were increased by 1.9- and 1.4-fold in Ubi1:AtJMT and drought-treated NT panicles, respectively. The ABA increase in Ubi1:AtJMT panicles grown in nondrought conditions suggests that MeJA, rather than drought stress, induces ABA biosynthesis under drought conditions. Using microarray and quantitative polymerase chain reaction analyses, we identified seven genes that were regulated in both Ubi1:AtJMT and drought-treated NT panicles. Two genes, OsJMT1 and OsSDR (for short-chain alcohol dehydrogenase), are involved in MeJA and ABA biosynthesis, respectively, in rice (Oryza sativa). Overall, our results suggest that plants produce MeJA during drought stress, which in turn stimulates the production of ABA, together leading to a loss of grain yield.

Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis.

AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 transcript accumulation within 30 min. The gene was also activated under various abiotic stresses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an AtMYB44 promoter-driven beta-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more sensitive to ABA and has a more rapid ABA-induced stomatal closure response than wild-type and atmyb44 knockout plants. Transgenic plants exhibited a reduced rate of water loss, as measured by the fresh-weight loss of detached shoots, and remarkably enhanced tolerance to drought and salt stress compared to wild-type plants. Microarray analysis and northern blots revealed that salt-induced activation of the genes that encode a group of serine/threonine protein phosphatases 2C (PP2Cs), such as ABI1, ABI2, AtPP2CA, HAB1, and HAB2, was diminished in transgenic plants overexpressing AtMYB44. By contrast, the atmyb44 knockout mutant line exhibited enhanced salt-induced expression of PP2C-encoding genes and reduced drought/salt stress tolerance compared to wild-type plants. Therefore, enhanced abiotic stress tolerance of transgenic Arabidopsis overexpressing AtMYB44 was conferred by reduced expression of genes encoding PP2Cs, which have been described as negative regulators of ABA signaling.

Overexpression of salicylic acid carboxyl methyltransferase reduces salicylic acid-mediated pathogen resistance in Arabidopsis thaliana.

We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomyces orontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants.