Organ-specific expression of brassinosteroid-biosynthetic genes and distribution of endogenous brassinosteroids in Arabidopsis

Abstract

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. There is only limited information on where BRs are synthesized and used. We studied the organ specificity of BR biosynthesis in Arabidopsis, using two different approaches: We analyzed the expression of BR-related genes using real-time quantitative reverse transcriptase-polymerase chain reaction, and analyzed endogenous BRs using gas chromatography-mass spectrometry. Before starting this study, we cloned the second BR-6-oxidase (BR6ox2) gene from Arabidopsis and found that the encoded enzyme has the same substrate specificity as the enzyme encoded by the previously isolated 6-oxidase gene (BR6ox1) of Arabidopsis. Endogenous BRs and the expression of BR-related genes were detected in all organs tested. The highest level of endogenous BRs and the highest expression of the BR6ox1, BR6ox2, and DWF4 genes were observed in apical shoots, which contain actively developing tissues. These genes are important in BR biosynthesis because they encode the rate-limiting or farthest downstream enzyme in the BR biosynthesis pathway. The second highest level of endogenous BRs and expression of BR6ox1 and DWF4 were observed in siliques, which contains actively developing embryos and seeds. These findings indicate that BRs are synthesized in all organs tested, but are most actively synthesized in young, actively developing organs. In contrast, synthesis was limited in mature organs. Our observations are consistent with the idea that BRs function as the growth-promoting hormone in plants.

Figure 1

BR biosynthesis pathway in Arabidopsis. The proposed biosynthetic pathway for BL from mevalonate (MVA) is shown with identified enzymes.

Figure 2

Sequence comparison of BR6ox2 and related genes. A, Sequence alignment of the BR6ox2, BR6ox1, and tomato Dwarf genes. Reverse contrast characters highlight identical amino acid residues and hatched characters indicate conserved residues. The GenBank/EMBL/DDBJ accession numbers of the BR6ox1 (CYP85A1) and the tomato Dwarf gene (CYP85A1) are AB035868 and U54770, respectively. The heme-binding signature sequence is underlined. B, Phylogenetic relationship between BR6ox2 and selected P450 genes. BR6ox2 belongs to a group consisting of Dwarf (tomato, CYP85A1), BR6ox1 (Arabidopsis, CYP85A1), CPD (Arabidopsis, CYP90A1 and X87367), ROT3 (Arabidopsis, CYP90C1 and AB008097), DWF4 (Arabidopsis, CYP90B1 and AF044216), and CYP90D (Arabidopsis, AB066286). BAS1/CYP72B1 (Arabidopsis, BAS1 and AC003105) is reported to be involved in BR inactivation. Dwarf3 (maize [Zea mays], CYP88A and U32579) and GA3 (Arabidopsis, CYP701A and AF047720) are reported to be involved in GA biosynthesis. The first P450 genes functionally identified from higher plants are CYP73A (Helianthus annuus cinnamate 4-hydroxylase, Z17369) and CYP75A (Petunia hybrida flavonoid-3′,5′-hydroxylase, D14588); both belong to the higher plant-specific group A of P450 genes. The accession numbers of the other members are D30718 (CYP8), M93133 (CYP7A), and X90458 (CYP86A).

Figure 3

Kinetics of regulation of the BR6ox2 gene by BL. Light-grown det2 seedlings were treated with 10 nm BL for the indicated times. Transcript abundance was analyzed using the Taq-Man RTQ RT-PCR. Transcript abundance levels are given as relative values normalized to 18S ribosomal RNA levels. Data are shown as the means ± se from three different plant samples.

Figure 4

The expression of BR-related genes in various organs. Arabidopsis seedlings were grown in soil for 4 to 12 weeks. The aerial parts of the plants were divided into apical shoots (As), inflorescence stems (St), rosette leaves (rL), cauline leaves (cL), and siliques (Si). Alternatively, plants were germinated on agar medium for 7 d, then cultured in liquid medium for 7 to 30 d, and then divided into shoots (Sh) and roots (Ro). Transcript levels were analyzed by the Taq-Man RTQ RT-PCR. Transcript levels are given as relative values to As (the value of 1), after being normalized to the 18S ribosomal RNA levels. Data are shown as the means with variation bars (sd) from three different plant samples for As, St, rL, Si, Sh, and Ro, or from two different plant samples for cL.