Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles ofBRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However,bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore,BRI1 is required for the homeostasis of endogenous BR levels.

Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating the phosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1 (BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and a GSK3 kinase, BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown how the perception of BR at the plasma membrane connects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmic shuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3 proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for 14-3-3 binding, another phosphorylation domain in BZR1 is required for the BIN2-induced nuclear export of BZR1. Mutations of putative phosphorylation sites in two distinct domains enhance the nuclear retention of BZR1 and BR responses in transgenic plants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22α-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22α-hydroxylase.

Summary: Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22α hydroxylase (CYP90B1) step that is the putative rate‐limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light‐ and dark‐grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws‐2 and AOD4 revealed that dwf4 accumulated the precursors of the 22α‐hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws‐2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild‐type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.

Brassinosteroids (BRs) are plant hormones that regulate growth and development. They share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination in plants has been only speculative. We show that BRs control sex determination in maize revealed through characterization of the classical dwarf mutant nana plant1 ( na1 ), which also feminizes male flowers. na1 plants carry a loss-of-function mutation in a DET2 homolog—a gene in the BR biosynthetic pathway. The mutant accumulates the DET2-specific substrate ( 24R )-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize plants with BR biosynthesis inhibitors completely mimicked both dwarf and tasselseed phenotypes of na1 mutants. Tissue-specific na1 expression in anthers throughout their development supports the hypothesis that BRs promote masculinity of the male inflorescence. These findings suggest that, in the monoecious plant maize, BRs have been coopted to perform a sex determination function not found in plants with bisexual flowers.