Rice lamina inclination

The Rice Lamina Inclination as a Specific Bioassay for Brassinosteroids... 

The isolation and structural determination of brassinolide [BR] was reported in Nature by Dr. N. B. Mandava and co-workers at the USDA in 1979(5), and was subsequently announced in Chem, Eng, News 6). The steroidal skeleton of the chemical structure of BR prompted us to examine the effect on the LJT. (225,235)-homobrassinolide was synthesized by Mori in 1980( 7) at our request just after the structural determination of BR. BR itself was also produced by Ikekawa and his co-workers the same year(3). Wada, a co-worker in our group, tested the activity of BR and homobrassinolide on rice lamina inclination and found that both compounds dramatically induced the inclination of laminae (see Table I) as expected(5). These findings strongly suggested... 

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). 

Rice lamina inclination test Radish seedling elongation Tomato hypocotyl elongation Wheat leaf-unrolling Pea inhibition ... 

Table L Biological Activity of Selected Brassinosteroid Analogs in the Rice Lamina Inclination Test...

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. 

Figure 2. Distribution of biological activity in HPLC on an ODS column. Biological activity is expressed as the response in the rice lamina inclination test...

Examination on the production of brassinosteroid-like active substances. The cells were extracted with methanol, and the NE fractions of the extracts were subjected to bioassay using the rice lamina inclination test by the same... 

Therefore, we examined the effect of 2,4-D added to the medium on production of brassinosteroids using V208 and V277 cells. However, addition of 10 ppm of 2,4-D to the culture medium at the start of the culture process caused growth inhibition of the crown gall cells. Therefore, 2,4-D was added to the medium when cultures were 10-days old. After culturing another 4 days, the cells were harvested and extracted with methanol. Brassinosteroid activity of the NE fractions of the cell extracts was examined by the rice lamina inclination test. The results are shown in Figure 8. 

Since the isolation of BL, BL and its related compounds have been tested by a number of bioassays originally designed for known plant hormones. BRs have been shown to have a broad spectrum of biological activities (3,4,8). Structure-activity relationship of BRs has also been clarified by bean second-intemode bioassay, bean first-intemode bioassay, raphanus test, tomato test, and rice-lamina inclination test (25-57). 

Fig. 3 Reversed-phase HPLC of the shoot and seed extracts of P. vulgaris as monitored by rice-lamina inclination test [(A) stem (C) seed] and RIA with anti-CS antiserum [(B) stem (D) seed]. Dosages in fresh-weight tissue equivalents 150 g in A 2 g in B 30 g in C and 0.5 g in D. TeS teasterone TyS typhas-terol.

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. 

Rice—Lamina Inclination, Endogenous Levels in Plant Tissues and Accumulation during Pollen Development of Brassinosteroids... 

Since the discovery of brassinolide, ten years ago, research on brassinolide and related steroids has attained a remarkable degree of development. So far, twenty-three brassinosteroids [BS] have been isolated and identified from twenty-two plant species. Furthermore, many unknown brassinolide-like active substances have been detected, so that the number of identified ones is likely to increase in the future. The background to successful isolation of new BS from various plant sources is due to the use of a rice-lamina inclination test for screening that is sensitive and specific for brassinosteroids. 

Brassinosteroid-induced rice-lamina inclination response. 

Brassinosteroid-induced Rice-lamina Inclination Response... 

We developed several methods for construction of the side chain and the A/B ring of brassinosteroids, and synthesized more than fifty analogs (6). In order to obtain the structure-activity relationship, those analogs were subjected to bioassay in the rice-lamina inclination (7), Raphanus, and tomato tests (5). In general, die structural... 

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