Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum

Abstract

An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC-MS analysis in both plants. In contrast, the polar fractions obtained from BR-deficient mutants, Arabidopsis cyp85a2 and tomato d(x) , did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants.

Identification of individual components of a commercial wheat germ acid phosphatase preparation

“…Commercial wheat germ acid phosphatase (WGAP) is extracted from the germ/embryo of wheat plants and is commonly used in vitro in many biochemistry laboratories. Specifically, its availability and inexpensive cost have historically made it a common choice for many projects which require a broad specificity phosphatase, including those in education and research (see, for example, references [2][3][4][5][6][7][8][9][10][11][12]). While the name wheat germ acid phosphatase implies a singular protein, it typically refers to a commercial preparation of partially purified protein which is known to contain multiple proteins [13][14][15][16][17][18].…”