Rice lamina inclination assay

A combination of the rice lamina inclination assay and GC/MS or GC/SIM analyses have allowed for identification of the brassinosteroids in various plant tissues These include brassinolide, 6-deoxodihydrocastasterone, brassinone from the insect galls of Castanea crenata (18,20) castasterone and 6-deoxodihydrocastasterone from the stems, leaves and flowers of Castanea crenata (20) brassinolide, castasterone, typhasterol (7), and teasterone (8) from leaves of Thea sinensis (21-23) castasterone and brassinone from the fruit of Pharbitis purpurea ( Japanese morning glory ) (24) brassinolide, castasterone and 24-epibrassinolide(29) from immature seed and/or pollen of Vida faba (broad bean ) (25,26) brassinolide and castasterone from the pollen of Alnus glutinosa (European alder) (27). 

The occurrence of brassinosteroids in gymnosperms has been reported from conifers. Yokota ef al. isolated typhasterol ( 2-deoxycastasterone ) from pollen of Japanese black pine ( Pinus thunbergii ) (38) and identified castasterone and typhasterol from shoots of sitka spruce (Picea sitchensis ) (39). Kim ef al. also identified castasterone and brassinolide from cambial scrapings of Scots pine (Pinus silverstris ), using a modified dwarf rice lamina inclination assay which showed a synergistic response of brassinosteroids with indole-3-acetic acid (40). 

Figure 3 shows hypothetical biosynthetic pathway of brassinolide. Namely campesterol or its analogs will be converted to teasterone via several steps of oxidation, then successively oxidized to typhasterol, castasterone and brassinolide. In parentheses, biological activities in the rice lamina inclination assay are shown. The... 

Biological activity was determined by the rice lamina inclination assay. HPLC support and mobile phase A, Cjg silica and 45% acetonitrile B, Cjg silica and 45% acetonitrile C, silica (Aquasil) and chloroform-methanol (95 5, 1% water). Abbreviations CS, castasterone BL, brassinolide. 

Relative biological activities in the rice lamina inclination assay are given in parentheses. 

The rice lamina inclination assay is very sensitive to brassinosteroids (11). In the second set of studies, we examined the metabolism of radioactive castasterone in the rice lamina assay (Yokota, T. et. al., unpublished data). The fate of tritiated castasterone was monitored for 72 hr. During incubation, again brassinolide was not detected. However, polar metabolites accumulated and the amount increased during 72 hr. (Figure 7). The polar metabolites seemed not to be changed after hydrolysis using either enzyme, hydrochloric acid or sodium hydroxide. 

Specificity. Using the first member of the family, brassinolide (BR), an extensive survey of its effects in 17 bioassays, which varied in their responses to gibberellins, auxins and cytokinins, showed that BR did not behave exclusively as any one of those hormones. In some supposedly specific bioassays BR was as effective, or more so, as the hormone the assay was supposed to detect (9,10). This also applies to the rice lamina inclination assay (11), which is now frequently used. 

Deoxocastasterone, an important intermediate in this pathway, is usually contained in plant tissues at the highest level [28,50]. However, this BR and some other 6-deoxoBRs have been considered to be end-pathway BRs because these show very low biological activity when examined by the rice lamina inclination assay [28]. In this bioassay, brassinolide promotes the bending of rice leaf joints at as low as 0.1 nM. Nonetheless... 

Overall, epimerization of the 3a-hydroxyl and/or introduction of a hydroxyl group at C25 and C26 is critical for the metabolism of 24-epicastasterone and 24-epibrassinolide in tomato cells. In the rice lamina inclination assay, 25-hydroxy-24-epibrassinolide is tenfold more active than 24-epibrassinolide, but 26-hydroxy-24-epibrassinolide is less active. However, 25-hydroxy-24-epibrassinolide is hundred-fold less active in restoring the dwarf phenotype of the BR-deficient Arabidopsis mutants [72], Therefore, although the... 

The physiological concentrations of BRs in plants are extremely low (ng.Kg Fw), and it can be veiy difficult to analyze their abundance in plant tissues. A wide range of methods are currently employed for the determination and quantification of brassinosteroids in plants, including bioassays, diverse chromatographic procedures, radioimmunoassays [9], and enzyme-linked immunosorbent assays [10-13]. The most widely used bioassays are the second bean intemode bioassay and rice-lamina inclination test. These bioassays have been used in the isolation of brassinolide from rape pollen [1] and castasterone from chestnut insect galls [14]. Gas chromatography-mass spectrometry (GC-MS) analysis is the current standard technique for instrumental analysis of BRs [15-17]. 

The development of bioassays for isolating bioactive compounds from natural sources has played an important role in recent smdies of namral BR phytochemistry. The development of highly sensitive and specific bioassays was essential for the isolation and purification of BRs from plant tissues because of the very low physiological concentrations of these hormones. The bean second intemode assay was used to isolate BL from rape pollen [1], and the rice-lamina inclination test was used to isolate CS from chestnut insect galls [14]. Following the publication of these results, the rice-lamina inclination test has been widely used to isolate many BRs from various plant sources because of its simplicity, high sensitivity, and specificity for BRs [25]. 

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