Levels Of Brassinolide Research Articles

PmLBD3 links auxin and brassinosteroid signalling pathways on dwarfism in Prunus mume

BackgroundGrafting with dwarf rootstock is an efficient method to control plant height in fruit production. However, the molecular mechanism remains unclear. Our previous study showed that plants with Prunus mume (mume) rootstock exhibited a considerable reduction in plant height, internode length, and number of nodes compared with Prunus persica (peach) rootstock. The present study aimed to investigate the mechanism behind the regulation of plant height by mume rootstocks through transcriptomic and metabolomic analyses with two grafting combinations, ‘Longyan/Mume’ and ‘Longyan/Peach’.ResultsThere was a significant decrease in brassinolide levels in plants that were grafted onto mume rootstocks. Plant hormone signal transduction and brassinolide production metabolism gene expression also changed significantly. Flavonoid levels, amino acid and fatty acid metabolites, and energy metabolism in dwarf plants decreased. There was a notable upregulation of PmLBD3 gene expression in plant specimens that were subjected to grafting onto mume rootstocks. Auxin signalling cues promoted PmARF3 transcription, which directly controlled this upregulation. Through its binding to PmBAS1 and PmSAUR36a gene promoters, PmLBD3 promoted endogenous brassinolide inactivation and inhibited cell proliferation.ConclusionsAuxin signalling and brassinolide levels are linked by PmLBD3. Our findings showed that PmLBD3 is a key transcription factor that regulates the balance of hormones through the auxin and brassinolide signalling pathways and causes dwarf plants in stone fruits.

Brassinolide as potential rescue agent for Pinellia ternata grown under microplastic condition: Insights into their modulatory role on photosynthesis, redox homeostasis, and AsA-GSH cycling

Microplastic (MP), as a new pollutant, not only affects the growth and development of plants but also may affect the secondary metabolites of plants. The anti-tumor role of Pinellia ternata is related to secondary metabolites. The role of brassinolide (BR) in regulating plant resistance is currently one of the research hotspots. The paper mainly explores the regulation of BR on growth and physiology of Pinellia ternata under MP stress. The experimental design includes two levels of MP (0, 1%) and two levels of BR (0, 0.1 mg/L). MP led to a marked reduction in plant height (15.0%), Fv/Fm (3.2%), SOD and APX activity (15.0%, 5.1%), whereas induced an evident raise in the rate of O2·- production (29.6%) and GSH content (4.4%), as well as flavonoids (6.8%), alkaloids (75%), and β-sitosterol (26.5%) contents. Under MP addition, BR supply significantly increased plant height (15.7%), aboveground and underground biomass (16.1%, 10.3%), carotenoid and GSH content (11.8%, 4.2%), Fv/Fm (2.9%), and activities of SOD, GR, and MDHAR (32.2%, 21.08%, 20.9%). These results indicate that MP suppresses the growth of P. ternata, although it promotes secondary metabolism. BR can alleviate the inhibitory effect of MP on growth by improving photosynthesis, redox homeostasis, and the AsA-GSH cycle.

PscCYP716A1-Mediated Brassinolide Biosynthesis Increases Cadmium Tolerance and Enrichment in Poplar.

Cadmium (Cd), as one of the heavy metals with biological poisonousness, seriously suppresses plant growth and does harm to human health. Hence, phytoremediation was proposed to mitigate the negative effects from Cd and restore contaminated soil. However, the internal mechanisms of detoxification of Cd used in phytoremediation are not completely revealed. In this study, we cloned the cytochrome P450 gene PscCYP716A1 from hybrid poplar “Chuanxiang No. 1” and found that the PscCYP716A1 was transcriptionally upregulated by Cd stress and downregulated by the exogenous brassinolide (BR). Meanwhile, PscCYP716A1 significantly promoted the poplar growth and enhanced the Cd accumulation in poplar. Compared to wild-type poplars, overexpressed PscCYP716A1 lines produced higher levels of endogenous BR and showed a stronger tolerance to Cd, which revealed that PscCYP716A1 may reduce the oxidative stress damage induced by Cd stress through accelerating BR synthesis. In general, PscCYP716A1 has a potential superiority in regulating the plant's tolerance to Cd stress, which will provide a scientific basis and a new type of gene-modified poplar for Cd-pollution remediation.

Effects of exogenous melatonin on plant growth, root hormones and photosynthetic characteristics of trifoliate orange subjected to salt stress

Soil salinity has negative effects on crop growth and production, and melatonin (MT) plays an important role in regulating plant salt stress. However, it is not clear whether exogenous melatonin mitigates the negative effect on citrus plants subjected to salt stress. This study aimed to explore the response of exogenous melatonin (0, 50, 100, 150 µmol/L) on plant growth, root hormone levels and the photosynthetic system of trifoliate orange (Citrus (Poncirus) trifoliata L.) seedlings exposed to 0 or 150 mmol/L NaCl for 4 weeks. The 150 mmol/L NaCl treatment significantly increased root zeatin riboside, gibberellin and brassinolide levels, while dramatically reducing plant growth, root auxin levels, leaf photosynthesis and fluorescence indexes of seedlings. However, melatonin treatment partially ameliorated reductions in plant height and dry matter accumulation caused by salt stress. Melatonin (100 µmol/L) appears to interact with IAA but not the other hormones studied. Furthermore, the effects of NaCl stress on the net photosynthetic rate, stomatal conductance, maximum photochemical efficiency, PSII effective photon yield, photochemical quenching and other indicators of seedlings leaves were also partially alleviated and the damage of NaCl stress was also reduced when seedlings were treated with melatonin. This suggests that 100 µmol/L melatonin may be an effective treatment.

Seed bulb size influences the effects of exogenous brassinolide on yield and quality of Pinellia ternata.

In recent years, natural Pinellia ternata populations of have gradually been exhausted, while the cultivated yield has been limited due to lack of research and uncertain climate condition. Therefore, it is necessary to explore methods of improving yield and quality in P. ternata using brassinolide (BR) treatments and choice of a suitable seed bulb size. This article reports the effects of BR and two seed bulb sizes (diameter: 0.5-1.0 cm and 1.0-1.5 cm) on active and nutrient components and antioxidant activity in P. ternata. The experiment included six levels of BR (0, 0.05, 0.10, 0.50, 1.00 and 2.00 mg l-1 ). The tuber yield of the two seed bulb sizes and bulbil yield of small seed bulbs increased 5.67%, 22.66% and 69.23% by day 105 after 0.50 mg l-1 BR treatment, compared with the control. On day 105, only 0.05 mg l-1 BR increased scores in principal components analysis (PCA) in tubers of small seed bulbs by 167.29%, and 0.05 and 0.50 mg l-1 BR increased PCA score in bulbils of large seed bulbs by 145.66% and 252.97%, respectively, compared with the control. Significant BR × seed bulb size interactions were found on yield and quality of P. ternata. The results indicate that BR effects on yield and quality of tubers and bulbils of P. ternata are not only related to BR concentration but also to seed bulb size.

Gibberellins, brassinolide, and ethylene signaling were involved in flower differentiation and development in Nelumbo nucifera

Hormones play important roles in vegetative and reproductive processes; however, the regulatory roles of hormones in Nelumbo nucifera (Lotus) growth and development are unclear. In this study, nine types of endogenous hormones, including gibberellins (GA1, GA3, and GA4), indole-3-acetic acid (IAA), brassinolide (BR), ethylene (ETH), jasmonic acid (JA), abscisic acid (ABA), and zeatin (ZT) were detected in dormant shoot tips, vegetative shoot tips, developing leaf buds, and developing flower buds of lotus. The results indicated that GA, ETH, and BR signaling can promote vegetative and reproductive development of lotus. GA signaling regulates plant height and stimulates flower bud differentiation. GA levels were the highest in the flower buds; exogenous GA3+4 increased plant height by approximately 90%, increased flower quantity by nearly 40%, and advanced flowering by 4 d. Suppressing GA biosynthesis using paclobutrazol decreased plant height and flower quantity by 38% and 87.1%, respectively, and delayed flowering by 15.6 d. ETH signaling has positive regulatory effects on vegetative growth and flower development. The ETH concentration in the developing leaf buds was at least 50% higher than that in other samples. Ethephon spraying led to remarkable increases in plant height and leaf thickness and extended the flowering duration. BR signaling acts as a growth promoter during vegetative and reproductive development in lotus. The highest BR levels were detected in the vegetative shoot tips. External application of 28-epihomobrassinolide resulted in growth-promoting phenotypes including longer scapes, thicker leaves, and prolonged flowering.

Plant Growth Regulator-Brassinolide for Mitigating Field Waterlogging Stress on Maize

Aim: To assess the pleiotropic role of a plant growth regulator, commercially identified as brassinolide (BR) in mitigating waterlogging stress imposed on maize.
 Study Design: A factorial combination of two maize varieties [Ikom White (IKW) and Oba-98], two BR levels (0 and 250 ml) and two waterlogging stages of maize growth [control (WL0) and seedling stage (WL1)], arranged as a split-split plot in a randomized complete block design with three replications was used.
 Place and Duration of Study: Akpabuyo Local Government Area, Cross River State-Nigeria. A two-year field experiment was conducted during the dry seasons of December 2016 and December 2017.
 Methodology: Waterlogging test was conducted on plots by demarcating them with 3.6 by 1.7 m metal sheets buried to a depth of 60 cm to prevent lateral soil-water movement. Two maize seeds were sown at 25 cm within and 75 cm between rows. The BR (250 ml) was sprayed foliar at 21 DAS. The non-waterlogging plots served as control. Observations were made on growth and yield variables as well as the plant's physiological traits.
 Results: Waterlogging significantly reduced the growth attributes of maize and increased (p≤0.05) the leaf moisture content. The photosynthetically active radiation on maize plants was substantially reduced (p≤0.05) by the waterlogging stress. Dry matter yield (DMY) and nutrient uptake in the leaves, stems and grains were reduced (p≤0.05) at both silking and at harvest. The effect of the BR was greater in Oba-98 with higher nutrient contents, radiation absorption, dry matter and grain yields than IKW.
 Conclusion: Treatment of maize plants with BR could induce some tolerance of field waterlogging. Thus, for optimum efficiency in maize production under stressed soil condition of waterlogging, it is recommended that the foliar spray of BR at the 250 ml per plant rate be considered.

Response of Two Fig (Ficus carica L.) Varieties after Receiving Brassinolide on Leaf, Shoot and Root Segment

Background and Objective: The increase in growth after receiving brassinolide may vary between species. The main growth factors which may directly reflect to yield are functional leaf, shoot and root segment. Thus, the aim of this study was to investigate the effect of different concentrations of exogenous application of BL on growth of leaves, shoots and roots segments of fig. Materials and Methods: The experiment was arranged as Split Plot Randomized Complete Block Design (SRCBD) with 4 replications. Two fig cultivars Improved BrownTurkey (IBT) and Masui Dauphine (MD) were considered as a main plot and four level (0, 50, 100 and 200 mL LG1) of BL concentration asa sub plot. Experiment was conducted in an open field at Ladang 15, Faculty of Agriculture, University Putra Malaysia Serdang, Selangor,Malaysia, from May-December, 2017. Data was recorded weekly and monthly. Results: The results showed that growth of fig was affected by brassinolide levels and cultivars. Application of 50-200 mL LG1 BL increased growth of fig on leaves, shoots and roots segments in weekly and monthly observations. There was significant difference treatment of brassinolide and cultivar alone on growth of fig. Inaverage, concentration of brassinolide at 200 ML LG1 resulted highest growth performance of fig. The highest growth value of interaction between brassinolide and fig variety was on treatment of IBT+200 ML LG1. Between the varieties, IBT showed higher growth than MD.Significant negative correlation was noted only on between RL with RAD. Conclusion: The results of this study indicated that growth of fig on leaves, shoots and roots segments was affected by brassinolide levels and cultivars.

Brassinolide effects on maize (Zea mays L.) growth and yield under waterlogged conditions

Our aim was to assess if Brassinolide (BR) could ameliorate stress caused by waterlogging on maize. Two BR levels (with and without), two maize varieties [Ikom White (IKW) and Obatanpa-98 (Oba-98)] and three growth stages [control (WLo), seedling stage (WL1), and tasseling stage (WL2)] were studied under waterlogging lasting 10 days. Maize growth and development were significantly (p ≤ .05) reduced by waterlogging stress under WL1 than WL2. Waterlogging stress at WL1 adversely affected (p ≤ .05) the protein and relative water contents. The nitrogen (N) content among the plant partitions (leaves, stems, and grains) were reduced (p ≤ .05) at both silking and harvest. The beneficial effect of BR was more pronounced in Oba-98 with higher protein contents, dry matter yield, N-uptake and harvest index than IKW. Oba-98 was also better yielding than IKW. Thus, in a waterlogged soil, treatment of maize plants with BR at WL1 could induce some tolerance.

Phenological traits of wheat response to different levels of humic acid and brassinolide

A field experiment was conducted to investigate phonological traits of wheat response to different levels of humic acid and brassinolide at New Developmental Farm The University of Agriculture, Peshawar during Rabi season 2009 -10. The wheat variety Saleem 2000 was sown. The design used was Randomized Complete Block Design (RCBD) with four replications in split plot arrangement. Humic acid was assigned to main plot whereas brassinolide was allotted to sub plot. Plot size of 3 x 3.5 m having 10 rows, 30 cm apart were used. Four levels of humic acid (0, 1, 2 and 4 kg ha) were applied at sowing times. Whereas 5 levels of brassinolide (0, 0.5, 1, 1.5 and 2 mg L) were sprayed in foliar form at the tillering stage. Application of 4 kg HA ha delayed days to heading (117.44). Increasing humic acid levels up to 2 kg ha delayed anthesis (124.18) days and maturity (156) days and produced taller (82.69) plants. Increasing brassinolide concentrations up to 2 mg L delayed days to heading (118.25days), anthesis (124.63days) and maturity (158days). Taller (89.56cm) plants were produced through the application of 2 mg BL. Increasing brassinolide concentration up to 2 mg L as combined with 2 kg HA ha delayed days to anthesis. Application of 2 kg HA ha enhanced wheat phenology and thus is recommended for wheat cultivation in the agro climatic conditions of Peshawar for improved phonological traits. Application of 2 mg BL improved wheat phenology and thus is recommended for wheat cultivation in the agro climatic conditions of Peshawar for better phonological traits.

Arabidopsis PIZZA Has the Capacity to Acylate Brassinosteroids

Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of (14)C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apical meristems in Brassica napus microspore-derived embryos.

Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4×10−6 M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.

Effects of Brassinolide with Naphthalene Acetic Acid on the Formation of Adventitious Roots, Trichome-Like Roots and Calli from Cultured Tobacco Leaf Segments, and the Expression Patterns of CNT103

We treated cultured tobacco leaf segments with brassinolide (BL) and naphthalene acetic acid (NAA) and determined that optimum concentrations of NAA for adventitious root, trichome-like root, and calli formation were, respectively, 10−6, 10−5, and 10−4 M. In the adventitious root formation group, the number and length of adventitious roots were increased at lower concentrations of BL; however, they became trichome-like roots at higher levels of BL. The trichome-like root formation group showed better development when a low concentration of BL was added. However, at higher concentrations of BL, trichome-like root production was reduced, forming calli instead. In the calli formation group, more calli were formed at low BL concentrations and after persistent exposure to BL regardless of BL concentration, and the size of the leaf segments increased. The CNT103 gene, which is expressed at the root tips showed increased levels of expression at BL concentrations up to 10−9 M and decreased levels of expression at BL concentrations over 10−9 M in the adventitious roots, trichome-like roots, and calli formation groups.

Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco

When several concentrations of brassinolide (BL) were added to a shoot induction medium (SIM) that contained only BA, redifferentiation of adventitious shoots from tobacco leaf discs was unaffected at low BL levels (10-10~10-8 M), but was inhibited at higher concentrations. In comparison, when BL was applied without BA, only cell expansion occurred and no shoots formed. The determination time for shoot formation was shortened at low BL concentrations, but their formation was postponed (i.e., time was lengthened) at higher concentrations. Elongation of shoots incubated for 30 d was unaffected at low BL concentrations, but was inhibited as that amount increased.NTH1, a tobacco homeobox gene that is expressed in the central zone of the tobacco shoot apex, showed greater expression levels in the SIM over time, and its expression was stronger in media treated with low concentrations of BL compared with the SIM control at the same time point. Expression ofNTH1 was postponed at higher BL concentrations. In conclusion, at low concentrations, brassinolide has no effect on shoot formation. However, it inhibits their formation at high concentrations when cytokinin is included in the media.

Cell‐Free Conversion of Castasterone in Cultured Cells of Phaseolus vulgaris and Marchantia polymorpha.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Cell-free Conversion of Castasterone in Cultured Cells of Phaseolus vulgaris and Marchantia polymorpha

Brassinosteroids (BRs) are steroidal plant hormones, which are required for the normal growth and development of plants. Castasterone (CS) and brassinolide (BL) are the most frequently identified BRs in the plant kingdom. Feeding experiments using isotope-labeled substrates revealed that BL is biosynthesized from CS by 7-oxalactonation. However, many plants which possess a fair amount of CS contain very low levels of BL. In addition, in plants which can convert CS to BL, the conversion rate is extremely low, yielding high levels of CS in the plants. Because CS, as well as BL, is known to induce feedback regulation of earlier steps in BR biosynthesis pathways, accumulation of CS may limit or alter BR biosynthesis in plants. Therefore, endogenous levels of CS should be reduced, after BL production, to a level below that at which feedback regulation can occur. This is a difficult proposition, as little is yet known about the catabolism of CS. This dearth of available data prompted us to investigate the catabolism of CS in cultured Phaseolus vulgaris and Marchantia polymorpha cells, in which the presence of CS and BL and conversion of CS to BL have been demonstrated. Cultured cells (5 g) of P. vulgaris and M. polymorpha were homogenized and centrifuged at 8, 000 × g for 10 minutes, and the resulting supernatants were re-centrifuged at 20,000 × g for 30 min. Cold acetone was then added to the obtained supernatants (final volume 40%), and the acetone precipitates were re-suspended in 0.1 M Na phosphate buffer (pH 7.4) containing 1.5 mM 2-mercaptoethanol and 30% glycerol for crude enzyme solutions. Non-labeled CS and NADPH (4.8 mM) were added to the enzyme solutions as a substrate and a cofactor, respectively, to examine catabolism of CS in the plants. After incubating at 37 C for 30 minutes, the assay mixtures were extracted with ethyl acetate (1.2 mL × 3). The obtained ethyl acetate soluble fractions were loaded on a Sep-Pak C18 cartridge eluted with 50%, and 100% methanol (5 mL each). The 100% methanol fractions were further purified by reversed phase HPLC (Nova Pak, C18, 8 × 100 mm) and eluted with 40% acetonitrile at a flow rate of 1 mL min−1. Fractions were collected every min, and analyzed by a preparative TLC (Merck, HPTLC F254) developed with a 6 : 1 mixture of chloroform and methanol. Besides fraction 19-21, which contained CS (added as the substrate), fraction 13-15 exhibited a BR-like blue-purplish spot at Rf 0.30. The metabolite in the fractions was analyzed by GC-MS/-SIM after methaneboronation. In GC-MS, bismethaneboronate (BMB) of the metabolite showed a molecular ion at m/z 498 and the most abundant ion at m/z 141, due to the fission of C20/C22, which was reduced in mass compared with CS BMB. The mass reduction suggests that a methyl in CS was eliminated in the metabolite. Therefore, the metabolite was proposed to be either 26-norCS or 28-norCS. In GC-MS, BMB of 26-norCS and 28-norCS showed basically the same mass spectrum, but their retention times on GC were clearly different. As shown in Table 1, GC retention time of BMB of the metabolite was equal to that of 26-norCS BMB. Consequently, the metabolite was characterized as 26-norCS. 26-NorCS showed approximately one-tenth the level of activity of CS, indicating that 26-norCS is a catabolite of CS in plant cells. Because isotope-labeled 26-norCS is not available, activity for the enzyme catalyzing the conversion of CS to 26-norCS, namely CS C-26 demethylase, was measured by GC-SIM based quantification method, yielding 0.90 and 0.31 ng mg−1

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea.

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.