PDF | Brassinosteroids are new and unique class of plant growth regulators that constitutes the sixth class of tinct from systemic acquired resistance (SAR) and and peroxidase activity in excised red cabbage. (Brassica. Cabbage Brassica campestris var. pekinensis. It has been . brassinosteroids and their structure activity relationship in the green alga Chlorella vulgaris. The optimal structure for highest BR activity normally is that found in BL, Effects of brassinosteroid on cell division and colony formation of Chinese cabbage.
This compound suffers dehydrogenation to 5-dehydroepisterol and hydrogenation to methylenecholesterol Isomerization of the D24 28 double bond to D24 25 produces methyldesmosterolthat is reduced to campesterol Oxidation of this sterol to 24R methylcholestenone is followed by saturation of the olefinic double bond to 24R -methyl-5a-cholestanone and reduction of the carbonylic function for campestanol 88 production.
The 14a-demethylation of obtusifoliol 95 to 4a-methyl-5a-ergosta-8,14,24 28 -trien-3b-ol 96 is encoded by a CYP51 enzyme steroid 14a-demethylase and Arabidopsis antisense AtCYP51 transgenic plants showed dwarfism during early development, slow growth during maturation, a high obtusifoliol 95 content but did not show phytosterol deficiency Kushiro et al. In the Arabidopsis fackel mutant seedlings was observed a high content of 96 and D8,unsaturated sterols was observed, and the wild phenotype was not rescued by brassinosteroid application, indicating the blockage of the conversion of 96 to 4a-methylfecosterol 97; Jang et al.
Two Arabidopsis mutants, dwf7 and ste1, were recognized to be unable to perform the conversion of episterol 99 to 5-dehydroepisteroland a third one, dwf5, is blocked in the conversion of 5-dehydroepisterol to methylenecholesterol The Arabidopsis dwf1 mutant is defective in the conversion of the last compound to campesterol The mutants dim and cbb1 are defective in the conversion of methyldesmosterol to campesterol The Arabidopsis mutants det2 and dwf6 are defective in the reduction of 24R methylcholestenone to 24R -methyl-5a-cholestanone The blocked biosynthetic step in the garden pea lkb mutant is the conversion of methylenecholesterol to campesterol 87; Nomura et al.
A small number of brassinosteroid biosynthesis mutants were recognized in the steps between campestanol 88 and brassinolide 1; figure In the Arabidopsis dwf4 mutant the conversions of campestanol 88 to 6-deoxocathasterone 54 and of 6-oxocampestanol 90 to cathasterone 53 are blocked, indicating that both substrates 88 and 90 are recognized by the same 22a-hydroxylase Choe et al.
In the Arabidopsis mutants cpd Szekeres et al. The tomato dpy mutant, an intermediate dwarf with severely altered morphology, is rescued by spraying with 6-deoxoteasterone 43 and subsequent precursors of brassinolide 1 in the late C-6 oxidation pathway, b ut not by 6-deoxocathasterone 54cathasterone 53 or their precursors Clouse and Feldmann, In tomato the late C-6 oxidation pathway seems to be the major route in brassinolide biosynthesis.
Analysis of the brassinosteroid fraction in the extreme dwarf dx tomato mutant showed that brassinolide biosynthesis is blocked in the conversion of 6-deoxocastasterone 36 to castasterone 9; Bishop et al. The conversion of teasterone 26 to typhasterol 25 and then to castasterone 9 was also observed in cultured cells of Marchantia polymorpha Park et al. Although this is evidence that the pathways depicted in figure 10 are common for brassinolide 1 biosynthesis in plant species other than Arabidopsis, pea and tomato Nomura et al.
In the case of norbrassinosteroids one would expect that they could be derived from cholesterolin a series of reactions similar to those occurring from campesterol Metabolic experiments with deuterium labeled castasterone 9 in Arabidopsis, rice, tomato and periwinkle detected norcastasterone 13 as a catabolite of castasterone 9; Fujioka et al.
The detection of nortyphasterol 30 in Arabidopsis Fujioka et al. Feeding experiments with labeled campestanol 88cholestanol 88a and cholesterol in Arabidopsis, tobacco and periwinkle revealed that cholesterol is converted to 4-cholestenone acholestanol 88a and 6-oxo-cholestanol 90abut the conversion ratios of cholesterol to cholestanol 88a are much smaller than those of campestanol 88 to cholestanol 88a, Nakajima et al.
The brassinosteroid metabolism was mainly studied in cultured cells of tomato and serradella using the corresponding 5,7,7-tris-tritiated brassinosteroids Kolbe et al. Cell suspension cultures of tomato convert 3-dehydroepiteasteronea putative precursor of epibrassinolide 6to epiteasterone and epityphasterol ; Kolbe et al. The enzymatic conversion of epiteasterone to 3-dehydroepiteasterone was monitored in cytosolic tomato and Arabidopsis thaliana fractions using fluorescent tagging and HPLC analysis.
In tomato, epicastasterone 14 is hydroxylated and glucosylated at C or C, yielding andor is dehydrogenated to 3-dehydroepicastasteronethat is reduced to 3,diepicastasterone This compound can be glucopyranosylated at C-2 or C-3 yielding and or hydroxylated at C resulting in hydroxy-3,diepicastasterone ; Hai et al. These hydroxylations are performed by two distinct enzymes, and hydroxylase proved to be a cytochrome P protein, while the hydroxylase seems to be a flavin-containing monooxygenase Winter et al.
It was detected that serradella Ornithopus sativus Brot. Trihydroxyketone is formed via transformation of epicastasterone 14 to 3,diepicastasterone 19which is oxidized to 20R-hydroxy-3,diepicastasterone and further to the pregnanedione followed by reduction figure In the same system, epibrassinolide 6; figure 16 was transformed into 2a,3b-dihydroxy-B-homooxa-5a-pregnane-6,dione ; Kolbe et al. A purified recombinant Brassica napus steroid sulfotransferase expressed by Escherichia coli catalyses the enzymatic brassinosteroid and precursor sulfonation specifically at position 22, as exemplified in figure 17 Rouleau et al.
It exhibited highest affinity for epicathasteronefollowed by epiteasterone In first experiments on microbial transformations of brassinosteroid incubation of epibrassinolide 6 with the fungus Cunninghamella echinulata yielded 12b-hydroxyepibrassinolide and the same 12b-hydroxylation was also observed with epicastasterone 14; Voigt et al.
On the other hand, the fungus Cochliobolus lunatus transformed epicastasterone 14 to the corresponding 15b-hydroxylated compound Voigt et al. The analogue 2a,3a-dihydroxyketocholestanewhen incubated with the fungus Mycobacterium vaccae, yielded 2a,3a,6a-trihydroxy-5a-androstaneone and 2a-hydroxyandrostene-3,dione ; Vorbrodt et al. Biological activity on insects Brassinosteroids show striking structural similarities with arthropod hormones of the ecdysteroid type such as hydroxyecdysone ; Adler and Grebenok, ; Lafontwhich led to several studies on the bioactivity of brassinosteroids and analogues on insects.
Inhibiting and antiecdysone effects have been observed in the course of such investigations Richter and Koolman, Thus, from a series of tested compounds castasterone 9 and 22,diepihomobrassinolidea synthetic 22b,23b-stereoisomer of homobrassinolide 2inhibited the evagination of imaginal disks of the Phormia terra-novae fly Hetru et al.
The 22,diepihomocastasterone analogue and lactoneto a lesser extent, bound competitively to ecdysteroid receptors from larvae of the Calliphora vicina blowfly representing first antiecdysones Lehmann et al.
Compounds and were shown to act as weak inhibitors of binding of the ecdysteroid ponasterone A binding to the intracellular ecdysteroid receptor from the epithelial cell line from Chironimus tentans and gave morphological effects and inhibition of chitin synthesis similar to the moulting hormones Spindler et al. Compound also exhibited a binding affinity to an ecdysteroid receptor in last instar larvae of the Galleria mellonella wax moth similar to ecdysone Sobek et al.
In the Drosophila melongaster II cell bioassay natural brassinosteroids showed no agonistic or antagonist activity Dinan et al. In other studies the cockroach Periplaneta americana has been used as preferred model.
In feeding experiments 22,diepihomobrassinolide created moulting retardation by about 11 days with the highest applied doses Richter et al. Similarly to the effects of the hormone hydroxyecdysone dose-dependent neurodepressing effects with compounds and, to a lesser extent, compound were observed on Periplaneta americana indicating an ecdysteroid agonistic activity Richter and Adam, Also the first evidence for a metabolic transformation of a brassinosteroid in insects has been shown recently with this species Schmidt et al.
Thus, an organspecific epimerization of the brassinosteroid to 2,diepicastasterone ; figure 20 could be detected in female insects when epicastasterone 14 was fed to the cockroach. The metabolite was observed only in the ovaries but not in the testes of the insect and was identified by GC-MS comparison with a synthesized sample Voigt et al.
The above-mentioned results indicate a series of biological effects of brassinosteroids on insects including in vitro cell culture and in vivo whole larvae. More detailed biological and biochemical studies using the structural multitude of brassinosteroids are necessary and could lead to new strategies to influence ecdysteroid-dependent steps of insect development and new pathways for insect pest control.
Biological activity and structure-activity relationships The biological activity of brassinosteroids was initially evaluated by the bean second internode assay Grove et al. In this test auxins and cytokinins are not detected and gibberellins elongate the treated and upper internodes. Brassinosteroids promote cell division and elongation, swelling, curvature and splitting of the treated internode: The bean first internode assay, used for evaluating the auxin-induced growth, was also employed for testing the structure-activity relationships of brassinosteroids Thompson et al.
The rice lamina inclination assay, based on a test originally developed for auxins Maeda,was modified for brassinosteroid detection Wada et al. While this assay has a limit of detection of 50ppm for indolacetic acid, the limit is 0. A modification, employing rice lamina of the whole seedlings pre-treated with IAA, diminished the limit of brassinolide 1 detection to 0. This test is considered as specific for brassinosteroids and is employed to detect and follow the purification of these natural products Takatsuto, ; Adam et al.
The wheat leaf unrolling bioassay, introduced inresponds to brassinolide 1 and castasterone 9 at a limit of detection of 0. Other assays are less frequently employed to evaluate brassinosteroids structure-activity relationships, such as the mung bean epicotyl elongation assay Gregory and Mandava,the radish Takatsuto et al.
Although the above biological assays are not equivalent, they allowed the establishment of relatively safe structural activity relationships Adam and Marquardt, ; Singh and Bhardwaj, ; Mandava, ; Abreu,with the aid of a series of brassinosteroid analogues.
As a general rule, the most bioactive brassinosteroids are of the 6-oxooxalactone type, followed by the 6-keto brassinosteroids and the 6-deoxo brassinosteroids that are almost inactive Mandava, Transforming 6-oxooxalactone to ether, thialactone, lactam, 6-oxaoxolactone, 6-azaoxalactone and 6-azathiolactone Okada and Mori, a; Kishi et al. The introduction of a hydroxyl at 5a decreases the brassinolide activity ca.
A less dramatic decrease in bioactivity on the rice lamina inclination assay has also been reported when a 5a-hydroxyl function is introduced on homocastasterone 10; Brosa et al. Within two years brasssinolide BL and its stereo isomer, epiBL, had been chemically synthesized, eliminating the need for such massive plant extraction procedures.
With ample synthetic compound in hand, research in the 's focused on determination of BR physiological effects in a wide variety of biological systems and on testing greenhouse and field applications for enhanced crop yield Cutler et al. The first report of the effects of BR on Arabidopsis growth appeared in Clouse and Zurek, followed shortly thereafter by a description of a screen for BR-insensitive mutants in Arabidopsis and demonstration that BRs regulated gene expression in that species Clouse et al.
The early 's also saw significant progress by several Japanese groups in unraveling the biosynthetic pathway to BRs from common membrane sterols Fujioka and Sakurai, While most chemists and biologists involved in BR research believed these compounds were indeed a new class of plant hormone Sasse,unequivocal proof of their indispensable role in plant growth and development was not available untilwhen a series of four independent reports described the identification and properties of one BR-insensitive Clouse et al.
The mutants exhibited an extreme dwarf phenotype, which could be rescued to wildtype by BR treatment of the deficient mutants. Moreover, it was demonstrated that two of the deficient mutants resulted from lesions in genes encoding steroid biosynthetic enzymes.
Materials and Methods Seeds of Brassica oleracea var. Seeds were surface sterilized with hypochlorite and rinsed th times with DDW and primed with epiBL and homoBL concentrations 0, M for 8th hours and DW as control.
The treated seeds were sown in three replications. Morphological data in terms of seedling growth germination, shoot length, root length, fresh weight and dry weight were measured on 10th day excluding germination it was observed on 3rd and 4th day. The samples were harvested for the various biochemical analyses 10 DAS. Determination of pigments Chl a, b and carotenoids Total chlorophyll Chlchlorophyll a, b and carotenoid Car content were quantified by following the method of Lichtenthaler et al.
Brassinosteroid phytohormones: structure, bioactivity and applications
Fresh leaves were homogenized in 3. Determination of carbohydrate, sugar, reducing and reducing sugar content Carbohydrate content was estimated by the method of Dubois et al. Reaction mixture contains phenol reagents, H2SO4 and samples were incubated at room temperature for 30 minutes.
Absorbance was taken at nm and amount of carbohydrate was expressed as mg g-1 fresh weight. Total sugar was estimated following the method of Loewus [ 10 ].
Reaction mixture contains 0. Reaction mixture was heated for 10 min in boiling water bath and cooled rapidly at room temperature. Absorbance was measured at nm. Amount of total sugars content was calculated and expressed as mg g-1 fresh weight.
Reducing sugar content was estimated by the method of Nelson [ 11 ]. Determination of protein content Protein estimation was done following the method of Lowry et al. Statistical analysis All analyses were done on a completely randomized design. Maximum germination recorded with 10—9 mM epiBL Fresh weight and dry weight were evaluated and var.
A similar trend in increase in fresh weight and dry weight was revealed in var. Increase in shoot length was recorded in all the primed treated varieties to control and effective amelioration in length was seen in 10—6 mM homoBL in var.
The experimental data revealed maximum root length in control in all the experimental varieties. Inhibition of root length in BRs primed seeds was observed in all the treatments of brassinosteroids. Brassinosteroids treatment significantly influenced the photosynthetic pigments.