CPD Gene Research Articles

Novel 4-chlorophenoxyacetate dioxygenase-mediated phenoxyalkanoic acid herbicides initial catabolism in Cupriavidus sp. DL-D2

Microbial metabolism is an important driving force for the elimination of 4-chlorophenoxyacetic acid residues in the environment. The α-Ketoglutarate-dependent dioxygenase (TfdA) or 2,4-D oxygenase (CadAB) catalyzes the cleavage of the aryl ether bond of 4-chlorophenoxyacetic acid to 4-chlorophenol, which is one of the important pathways for the initial metabolism of 4-chlorophenoxyacetic acid by microorganisms. However, strain Cupriavidus sp. DL-D2 could utilize 4-chlorophenoxyacetic acid but not 4-chlorophenol for growth. This scarcely studied degradation pathway may involve novel enzymes that has not yet been characterized. Here, a gene cluster (designated cpd) responsible for the catabolism of 4-chlorophenoxyacetic acid in strain DL-D2 was cloned and identified, and the dioxygenase CpdA/CpdB responsible for the initial degradation of 4-chlorophenoxyacetic acid was successfully expressed, which could catalyze the conversion of 4-chlorphenoxyacetic acid to 4-chlorocatechol. Then, an aromatic cleavage enzyme CpdC further converts 4-chlorocatechol into 3-chloromuconate. The results of substrate degradation experiments showed that CpdA/CpdB could also degrade 3-chlorophenoxyacetic acid and phenoxyacetic acid, and homologous cpd gene clusters were widely discovered in microbial genomes. Our findings revealed a novel degradation mechanism of 4-chlorophenoxyacetic acid at the molecular level.

Molecular Characterization of Enterococcus faecalis and Enterococcus faecium Isolated from a Meat Source in Shahrekord Local Markets, Iran.

Enterococcus faecalis (E. faecalis) and E. faecium (E. faecium) are commensals of the gastrointestinal biota of humans and animals and are considered opportunistic pathogens. This study aimed to improve the knowledge about E. faecalis and E. faecium isolated from meat. To this end, 104 meat samples were collected from sheep, goats, cattle, and calves from Shahrekord local markets in Iran. Presumptive colonies of E. faecalis and E. faecium were isolated from the samples and subjected to identification tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was performed to detect some virulence genes. Furthermore, randomly amplified polymorphic DNA typing and repetitive element sequence-based PCR fingerprinting were used to show the clonal relatedness of the isolates. The results revealed that enterococci were present in 90 out of 104 samples. Specifically, E. faecalis and E. faecium were the commonly isolated species, with the predominance of E. faecalis, which exhibited high resistance to streptomycin (95%) but was susceptible to vancomycin (85.6%). Virulence genes detection showed that ccf and cpd genes were the most prevalent genes in both species. In addition, the molecular typing method indicated that the isolates belonged to separate subgroups. This study shows the contamination of meat products by potential pathogens and resistant enterococci. There is a need to implement regular surveillance to monitor the emergence of antimicrobial-resistant E. faecalis and E. faeciumin food, particularly in meat production.

Wheat Type One Protein Phosphatase Participates in the Brassinosteroid Control of Root Growth via Activation of BES1.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

Small molecule inhibitors of mammalian GSK-3β promote in vitro plant cell reprogramming and somatic embryogenesis in crop and forest species.

Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. However, in many crop and forest species, somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, a representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis genes FUS3, LEC2, and AGL15. Plant GSK-3 kinase BIN2 is a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using GSK-3β inhibitors that could be extended to other species.

Functional Characterization of the Steroid Reductase Genes GmDET2a and GmDET2b form Glycine max.

Brassinosteroids are important phytohormones for plant growth and development. In soybean (Glycine max), BR receptors have been identified, but the genes encoding BR biosynthesis-related enzymes remain poorly understood. Here, we found that the soybean genome encodes eight steroid reductases (GmDET2a to GmDET2h). Phylogenetic analysis grouped 105 steroid reductases from moss, fern and higher plants into five subgroups and indicated that the steroid reductase family has experienced purifying selection. GmDET2a and GmDET2b, homologs of the Arabidopsis thaliana steroid -reductase AtDET2, are proteins of 263 amino acids. Ectopic expression of GmDET2a and GmDET2b rescued the defects of the Atdet2-1 mutant in both darkness and light. Compared to the mutant, the hypocotyl length and plant height of the transgenic lines GmDET2a and GmDET2b increased significantly, in both darkness and light, and the transcript levels of the BR biosynthesis-related genes CPD, DWF4, BR6ox-1 and BR6ox-2 were downregulated in GmDET2aOX-23 and GmDET2bOX-16 lines compared to that in Atdet2-1. Quantitative real-time PCR revealed that GmDET2a and GmDET2b are ubiquitously expressed in all tested soybean organs, including roots, leaves and hypocotyls. Moreover, epibrassinosteroid negatively regulated GmDET2a and GmDET2b expression. Sulfate deficiency downregulated GmDET2a in leaves and GmDET2b in leaves and roots; by contrast, phosphate deficiency upregulated GmDET2b in roots and leaves. Taken together, our results revealed that GmDET2a and GmDET2b function as steroid reductases.

Arabidopsis G-Protein β Subunit AGB1 Interacts with BES1 to Regulate Brassinosteroid Signaling and Cell Elongation.

In Arabidopsis, brassinosteroids (BR) are major growth-promoting hormones, which integrate with the heterotrimeric guanine nucleotide-binding protein (G-protein) signals and cooperatively modulate cell division and elongation. However, the mechanisms of interaction between BR and G-protein are not well understood. Here, we show that the G-protein β subunit AGB1 directly interacts with the BR transcription factor BES1 in vitro and in vivo. An AGB1-null mutant, agb1-2, displays BR hyposensitivity and brassinazole (BRZ, BR biosynthesis inhibitor) hypersensitivity, which suggests that AGB1 positively mediates the BR signaling pathway. Moreover, we demonstrate that AGB1 synergistically regulates expression of BES1 target genes, including the BR biosynthesis genes CPD and DWF4 and the SAUR family genes required for promoting cell elongation. Further, Western blot analysis of BES1 phosphorylation states indicates that the interaction between AGB1 and BES1 alters the phosphorylation status of BES1 and increases the ratio of dephosphorylated to phosphorylated BES1, which leads to accumulation of dephosphorylated BES1 in the nucleus. Finally, AGB1 promotes BES1 binding to BR target genes and stimulates the transcriptional activity of BES1. Taken together, our results demonstrate that AGB1 positively regulates cell elongation by affecting the phosphorylation status and transcriptional activity of BES1.

Effects of heterologous expression of Populus euphratica brassinosteroids biosynthetic enzyme genes CPD (PeCPD) and DWF4 (PeDWF4) on tissue dedifferentiation and growth of Arabidopsis thaliana seedlings

To understand the functions of Populus euphratica CPD (PeCPD) and DWF4 (PeDWF4), the responses to exogenous phytohormone in Arabidopsis-PeCPD and -PeDWF4 transgenic lines (PeCPD-TL and PeDWF4-TL) and corresponding wild type (WT) seedlings were investigated. Results showed that all of PeCPD-TL, PeDWF4-TL and WT seedlings cultured on the mediums containing 2,4-dichlorophenoxyacetic acid (2,4-D) + 6-benzylaminopurin (6-BA) or 2,4-D + 6-BA + brassinolide (BL) could be dedifferentiated to callus with 100% frequency, but they displayed strong differences in callus formation sites, callus growth rates (CGRs) and tissue dedifferentiation degrees. On the medium containing 2,4-D alone, the seedlings of all the plants could formed callus, but callus formation times (CFTs) were delayed, and callus formation rates (CFRs) were differentially decreased. After adding lower concentrations of BL, their CFRs were all restored to 100%, but tissue dedifferentiation degrees were obviously lower than these on the mediums with 2,4-D + 6-BA or 2,4-D + 6-BA + BL. On the mediums containing 6-BA or 6-BA + BL, the seedlings of all the plants failed to produce callus. Semi-quantitative RT-PCR analysis also showed that the transcription levels of PeCPD, PeDWF4, AtCPD and AtDWF4 in PeCPD-TL, PeDWF4-TL and WT were evidently different. These results suggest that PeCPD and PeDWF4 play similar but not exactly the same roles in the regulation of callus morphogenesis of Arabidopsis seedlings, and that BL can partially replace the role of cytokinin to induce callus formation through interacting with auxin.

Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

Analysis of gene copy number alterations by multiplex ligation-dependent probe amplification in columnar cell lesions of the breast

Columnar cell lesions (CCLs) are possible precursors of breast cancer, but little is known about the role of breast cancer-related genes in the progression of CCL to invasive breast cancer. Gene copy numbers of 17 breast cancer-related genes were analyzed using Multiplex Ligation-dependent Probe Amplification (MLPA) in CCL (N = 28), ductal carcinoma in situ (DCIS) grade I likely originating from CCL (N = 5), and paired CCL (N = 14/28) with DCIS (N = 7) and/or invasive carcinoma (N = 13). The genes included were BIRC5, C11orf30, CCND1, CCNE1, CDH1, CPD, EGFR, ERBB2, ESR1, FGFR1, IKBKB, MAPT, MED1, MTDH, MYC, TOP2A and TRAF4. No high level gene amplifications were observed in CCL, but copy number gains were encountered for the C11orf30 (3/28), MYC, CPD, MTDH (2/28), and CCND1, CCNE1, ESR1 and TOP2A genes (1/28). In addition, CDH1 showed loss in 2/28 and TOP2A in 1/28 cases. CCLs with or without atypia exhibited comparable numbers of copy number changes (p = 0.312). Overall, the frequency of gene copy number changes increased from CCL towards DCIS and invasive carcinoma (p = 0.004). Also in the cases with synchronous lesions, the CCLs exhibited fewer copy number changes than the DCIS/invasive carcinomas. CCLs carry copy number changes of several known breast cancer-related genes, thereby substantiating their role in breast carcinogenesis. Among them, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of particular interest. Since the copy number changes observed were more prevalent in DCIS and invasive carcinoma than in CCL, the corresponding gene alterations may represent rather late occurring events in low nuclear grade breast carcinogenesis.

Secondary Transfer and Expression ofvanAinEnterococcus faeciumDerived from a Commensal Vancomycin-SusceptibleEnterococcus faeciumMulti-Component Food Isolate

We investigated the potential for vancomycin-susceptible Enterococcus (VSE) from multi-component salads to disseminate vanA from four clinical vancomycin-resistant enterococci to 14 streptogramin-resistant enterococci (SRE) of food and animal origin. Strains were selected from a previous study based on the presence of streptogramin susceptibility and/or vanA, vanB, vatD, vatE, agg, cpd, and gelE genes. Transconjugants were isolated on brain heart infusion agar containing vancomycin and selective antibiotics. Thirty-nine matings using a 1:10 donor-recipient ratio for filter and broth methods resulted in transfer of vanA between an agg(+)cpd(+)gelE(+) Enterococcus faecalis donor and an agg(-)cpd(-)gelE(-) streptogramin-susceptible Enterococcus faecium salad recipient at a frequency of 10(-8) per recipient by filter method. Secondary mating of the transconjugant with SR/VSE strains resulted in a two- to four-log-fold greater frequency of transfer. Reverse transcription-polymerase chain reaction revealed vanA RNA products in the transconjugant cultivated in nutrient broth and salad at 37°C in the presence and absence of recipient filtrate. This study demonstrated that native salad VSE disseminated vanA to SRE carrying agg, cpd, and/or gelE. An increase in transfer efficiency resulted from secondary conjugation using the native vancomycin- and streptogramin-susceptible salad strain as the donor.

Virulence Factors in Enterococci from Partridges (Alectoris rufa) Representing a Food Safety Problem

Forty-three Enterococcus isolates recovered from fecal samples of partridges, during the partridge hunting season, were studied for gelatinase and β-hemolysis activities. The presence of fsr-gelE genes and the cyl operon was studied by polymerase chain reaction and correlated with gelatinase and β-hemolysis production, respectively. In addition, genes encoding additional virulence factors (cpd, hyl, agg, esp, and ace) was also analyzed in all enterococci. Most of our Enterococcus faecalis isolates showed gelatinase activity (10 of 15 isolates), and this activity was not present in the other enterococcal species. All enterococci that showed gelatinase activity harbored the gelE and fsr genes. A large proportion of our isolates harbored genes of the cyl operon (19 of 43 isolates), although only 1 isolate contained the five cyl tested genes (E. faecalis), being the only one that expressed β-hemolysis. From the additional virulence factors (cpd, hyl, agg, esp, and ace), at least one virulence gene was detected in 13 of the 15 E. faecalis isolates, with cpd being the most frequently detected gene (9 isolates), followed by agg (5 isolates) and ace (4 isolates) genes. These virulence genes were not detected in the other enterococcal species with the exception of one E. faecium and E. casseliflavus isolate that harbored the hyl and cpd genes, respectively. Moreover, the esp gene was not detected in any of our isolates. In conclusion, this study showed the presence of virulence factors in enterococci of partridges and the possible transmission to humans through the food chain.

Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals

The high prevalence of Enterococcus faecalis in root canal treated teeth with post-treatment disease, as evidenced by both molecular and traditional culturing methods, suggests that this species may be a key player in endodontic treatment failure. This study aimed to detect virulence factors by phenotypic and western blotting tests, and virulence genes by PCR from 20 clinical strains of E. faecalis isolated from treated root canals of teeth with (10) or without (10) apical periodontitis. Moreover, genomic diversity of these strains was assessed by pulsed-field gel electrophoresis (PFGE) and rep-PCR. All 20 strains presented the gelE gene (gelatinase), but 10 of them did not hydrolyze gelatin. Seven of the 10 gelatinase-producing isolates were recovered from root canals with lesions, which suggests a role for this virulence factor in the pathogenesis of post-treatment disease. The esp gene was expressed only in cases where gelatinase production was negative. The other virulence genes were found in 90% ( efaA and ace genes), 45% ( agg gene) and 95% ( cpd gene) of the E. faecalis isolates. As for PFGE and rep-PCR, no specific clonal type of E. faecalis was found in association with teeth with or without disease, revealing the interindividual clonal diversity of endodontic infections.

Streptogramin resistance patterns and virulence determinants in vancomycin-susceptible enterococci isolated from multi-component deli salads

This study examined vancomycin-susceptible Enterococcus (VSE) from deli salads for streptogramin resistance and presence of cpd, agg and gelE genes. Fifteen VSE from retail salads were isolated for identification and antimicrobial susceptibility testing by MicroScan, Etest and agar diffusion. Clinical vancomycin-resistant Enterococcus (n = 32) and animal VSE (n = 17) were included for comparative purposes. Multiplex PCR was used to detect the following genes: agg, gelE, cpd, vatD, vatE and sodA. Results showed fewer streptogramin-susceptible Enterococcus faecium isolated from salad (1/6, 17%) and animals (6/10, 60%) than from clinical (26/29, 90%) sources. A low level of erythromycin susceptibility was detected among salad (2/6, 33%) and animal (3/10, 30%) Ent. faecium isolates. Food and animal VSE demonstrated similarities in antimicrobial resistance profiles. All Enterococcus faecalis carried one or more of the selected genes cpd (40%), gelE (33%) and agg (27%). The vatD or vatE genes were not detected in any of the isolates. Experiments demonstrated that streptogramin resistance and virulence genes agg, cpd and gelE are present in enterococci isolated from deli salads. This study provides useful information regarding streptogramin resistance and virulence determinants in enterococci from foods associated with multi-component ingredients.

An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.

A proteomics study of the mung bean epicotyl regulated by brassinosteroids under conditions of chilling stress

Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77% identity with the Arabidopsis CPD gene. It was strongly suppressed by chilling stress. This implies that exogenous treatment with BR could allow the plant to recover from the inhibited growth caused by chilling. In this study, we used proteomics to investigate whether the mung bean epicotyl can be regulated by brassinosteroids under conditions of chilling stress. Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24-epibrassinolde; 17 proteins down-regulated by this chilling were re-up-regulated. These up-regulated proteins are involved in methionine assimilation, ATP synthesis, cell wall construction and the stress response. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment with DL-methionine. This is the first proteome established for the mung bean species. The regulatory relationship between brassinosteroids and chilling conditions was investigated, and possible mechanisms are discussed herein.

A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17β-hydroxysteroid dehydrogenase (17β-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed.

Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to DWF4 protein in the brassinosteroid biosynthesis pathway.

Triadimefon (Bayleton), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14 alpha-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.

Analysis of carbohydrate metabolism of CPD antisense plants and the brassinosteroid‐deficient cbb1 mutant

AbstractBrassinosteroids (BRs) are essential regulators of growth and development. BR‐deficient mutants such as cpd/cbb3 and dwf4 display extreme dwarfism due to a failure in cell elongation. To avoid the severe pleiotropic effects caused by the extreme growth defect, transgenic Arabidopsis lines carrying a construct for antisense inhibition of CPD gene expression were established and subjected to physiological analysis. The CPD‐antisense (α‐CPD) lines display characteristic phenotypic alterations of BR‐deficient plants such as reduced stem and petiole growth, smaller leaves, and a slightly delayed development. The observed changes are intermediate between the corresponding loss‐of‐function mutant (cbb3) and wild‐type plants. In the present study, the primary carbon metabolism of the transgenic lines as well as the BR‐deficient cbb1 (dwf1‐6/dim) mutant was analysed. Gas exchange measurements indicated a reduced assimilatory capacity of the α‐CPD plants. Soil‐grown α‐CPD as well as cbb1 (dwf1‐6) mutant plants show a clear reduction in starch content. The metabolic alterations are accompanied by altered enzyme activities such as reduced invertase and cytosolic β‐amylase activity, and altered expression patterns of genes such as Atbfruct1, Asus1, and ct‐Bmy (encoding a cell wall invertase, sucrose synthase, and plastidic β‐amylase, respectively). The impaired carbon assimilation, as well as the altered enzyme activities and gene expression patterns in the α‐CPD and cbb1 (dwf1‐6) plants, demonstrate the necessity of normal CPD and DIM expression for proper carbon uptake and metabolism and may point to an essential function of BRs. The impaired growth of BR‐deficient plants may be (at least in part) due to reduced photosynthesis.

Selective Interaction of Triazole Derivatives with DWF4, a Cytochrome P450 Monooxygenase of the Brassinosteroid Biosynthetic Pathway, Correlates with Brassinosteroid Deficiency in Planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.