Characterisation of two brassinosteroid C-6 oxidase genes in pea

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of P450s, and to date, two CYP85As have been isolated in tomato, two in Arabidopsis, one in rice, and one in grape. We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea ( Pisum sativum L.). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke , that blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterised pea BR mutants ( lk , lka and lkb ), and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea. the effect of the lke mutation on BR biosynthesis, metabolism and response pathways, we also conducted PCR expression analysis of the BR genes, LKB , LK , PsCPD1 , PsCPD2 , PsDWF4 , PsBR6ox1 and PsBR6ox6 (BR biosynthesis); PsBAS1 (BR metabolism); and LKA (BR perception) genes in WT and lke plants. Results show and PsBR6ox6 ), metabolism ( PsBAS1 ) and perception ( LKA ) were determined in young expanding internodes of WT and lke plants that had 10-leaves fully expanded. All results were obtained from three independent replicates, each consisting of tissue from three separate plants. Note that gene expression values are relative, not absolute and comparisons of expression levels between different genes are not valid.

[1]  Ho Bang Kim,et al.  A double mutant for theCYP85A1 andCYP85A2 Genes ofArabidopsis exhibits a Brassinosteroid dwarf phenotype , 2005, Journal of Plant Biology.

[2]  T. Yokota.  Running head: Roles of Brassinosteroids in Pea Seed Growth and Germination , 2007 .

[3]  S. Fujioka,et al.  Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols. , 2006, The Plant journal : for cell and molecular biology.

[4]  M. Matsuoka,et al.  Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice , 2006, Nature Biotechnology.

[5]  S. Takatsuto,et al.  Arabidopsis CYP85A2, a Cytochrome P450, Mediates the Baeyer-Villiger Oxidation of Castasterone to Brassinolide in Brassinosteroid Biosynthesis , 2005, The Plant Cell Online.

[6]  Y. Kamiya,et al.  The Last Reaction Producing Brassinolide Is Catalyzed by Cytochrome P-450s, CYP85A3 in Tomato and CYP85A2 in Arabidopsis* , 2005, Journal of Biological Chemistry.

[7]  C. Beveridge,et al.  The Branching Gene RAMOSUS1 Mediates Interactions among Two Novel Signals and Auxin in Pea , 2005, The Plant Cell Online.

[8]  J. B. Reid,et al.  The brassinosteroid growth response in pea is not mediated by changes in gibberellin content , 2005, Planta.

[9]  J. B. Reid,et al.  Brassinosteroid Deficiency Due to Truncated Steroid 5α-Reductase Causes Dwarfism in the lk Mutant of Pea1 , 2004, Plant Physiology.

[10]  J. B. Reid,et al.  Brassinosteroids Do Not Undergo Long-Distance Transport in Pea. Implications for the Regulation of Endogenous Brassinosteroid Levels1 , 2004, Plant Physiology.

[11]  M. Pfaffl,et al.  Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations , 2004, Biotechnology Letters.

[12]  J. B. Reid,et al.  Molecular characterization of the brassinosteroid-deficient lkb mutant in pea , 2001, Plant Molecular Biology.

[13]  G. Bishop,et al.  Unique and overlapping expression patterns of Arabidopsis CYP85 genes involved in brassinosteroid C-6 oxidation , 2004, Plant Molecular Biology.

[14]  J. B. Reid,et al.  The LKA gene is a BRASSINOSTEROID INSENSITIVE 1 homolog of pea. , 2003, The Plant journal : for cell and molecular biology.

[15]  C. Rameau,et al.  DETERMINATE and LATE FLOWERING Are Two TERMINAL FLOWER1/CENTRORADIALIS Homologs That Control Two Distinct Phases of Flowering Initiation and Development in Pea Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015701. , 2003, The Plant Cell Online.

[16]  J. Ozga,et al.  Pollination-, Development-, and Auxin-Specific Regulation of Gibberellin 3β-Hydroxylase Gene Expression in Pea Fruit and Seeds1 , 2003, Plant Physiology.

[17]  S. Fujioka,et al.  Organ-Specific Expression of Brassinosteroid-Biosynthetic Genes and Distribution of Endogenous Brassinosteroids in Arabidopsis , 2003, Plant Physiology.

[18]  Yukihisa Shimada,et al.  Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. , 2002, The Plant journal : for cell and molecular biology.

[19]  S. Fujioka,et al.  Microarray Analysis of Brassinosteroid-Regulated Genes in Arabidopsis , 2002, Plant Physiology.

[20]  F. Nagy,et al.  Regulation of Transcript Levels of the Arabidopsis Cytochrome P450 Genes Involved in Brassinosteroid Biosynthesis1 , 2002, Plant Physiology.

[21]  S. Kikuchi,et al.  Isolation and Characterization of a Rice Dwarf Mutant with a Defect in Brassinosteroid Biosynthesis , 2002 .

[22]  J. B. Reid,et al.  Hormone levels and response during de-etiolation in pea , 2002, Planta.

[23]  Y. Kamiya,et al.  Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis. , 2001, Plant physiology.

[24]  Y. Kamiya,et al.  Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis. , 2001, Phytochemistry.

[25]  S. Takatsuto,et al.  Brassinosteroid/Sterol synthesis and plant growth as affected by lka and lkb mutations of Pea , 1999, Plant physiology.

[26]  Jonathan D. G. Jones,et al.  The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Bisseling,et al.  Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A. , 1998, The Plant journal : for cell and molecular biology.

[28]  K. Feldmann,et al.  The DWF4 Gene of Arabidopsis Encodes a Cytochrome P450 That Mediates Multiple 22α-Hydroxylation Steps in Brassinosteroid Biosynthesis , 1998, Plant Cell.

[29]  K. Feldmann,et al.  An Arabidopsis Brassinosteroid-Dependent Mutant Is Blocked in Cell Elongation , 1998, Plant Cell.

[30]  T. Yokota.  The structure, biosynthesis and function of brassinosteroids , 1997 .

[31]  J. B. Reid,et al.  Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea , 1997, Plant physiology.

[32]  Jonathan D. G. Jones,et al.  The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family. , 1996, The Plant cell.

[33]  D. Pompon,et al.  Yeast expression of animal and plant P450s in optimized redox environments. , 1996, Methods in enzymology.

[34]  J. B. Reid,et al.  Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid) , 1994, Plant physiology.

[35]  W. F. Thompson,et al.  Initial characterization of a pea mutant with light-independent photomorphogenesis. , 1992, The Plant cell.

[36]  J. B. Reid,et al.  Internode length in Pisum. Two further gibberellin‐insensitivity genes, lka and lkb , 1989 .