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First published online July 6, 2007; 10.1104/pp.107.100560 Plant Physiology 145:87-97 (2007) © 2007 American Society of Plant Biologists
A Putative Hydroxysteroid Dehydrogenase Involved in Regulating Plant Growth and Development1Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Canada S7N 0W9 (F.L., X.W., E.W.T.T., A.J.C.); and Plant Functions Lab, Institute of Physical and Chemical Research, 2–1 Hirosawa, Wako, Saitama 351–0198, Japan (T.A.)
We have functionally characterized an Arabidopsis (Arabidopsis thaliana) gene AtHSD1 (At5g50600) that encodes a protein with homology to animal 11-  -hydroxysteroid dehydrogenase (HSD). Transgenic Arabidopsis plants overexpressing AtHSD1 (designated AOHSD plants) under the control of the cauliflower mosaic virus 35S promoter showed increased growth and seed yield as well as increased tolerance of saline stress and reduced seed dormancy. In canola (Brassica napus), transgenic plants overexpressing AtHSD1 also outgrew wild-type plants. AOHSD phenotypes were similar to those of plants that overproduced brassinosteroids (BRs) or overexpressed the BR receptor gene BRI1. A loss-of-function hsd mutant produced by RNA interference displayed a semidwarfed phenotype with reduced sensitivity to BRs. In contrast, AOHSD plants were hypersensitive to BRs and exhibited increased catabolism of abscisic acid (ABA). Germination of AOHSD seeds was less sensitive to ABA, while hsd seed was more sensitive to ABA during germination. AtHSD transcription was rapidly induced by BR treatment in wild type and was expressed widely in aerial plant parts, especially vascular tissues. This study demonstrates that AtHSD1 is involved in regulating growth and development in plants and is likely to promote or mediate BR effects. The gene has significant potential for improving growth and yield of canola and other agricultural crops.
Glucocorticoids are a class of animal steroid hormones involved in metabolic, inflammatory, cardiovascular, and behavioral processes as well as response to stress (Tomlinson et al., 2004  ). The enzyme 11--hydroxysteroid dehydrogenase (HSD) is a key regulator of the level of glucocorticoids and catalyzes the interconversion of biologically active glucocorticoid (cortisol in human and corticosterone in rats and mice) and inactive glucocorticoid (cortisone and 11-dehydrocorticosterone; Kallberg et al., 2002; McCormick et al., 2006). Although 11-hydroxy steroids have not (to our knowledge) been identified in plants, genome sequence annotation has identified eight HSD-like genes in Arabidopsis (Arabidopsis thaliana) whose functions have not been fully characterized.
We initially identified canola (Brassica napus) HSD as being highly expressed in nongerminating, abscisic acid (ABA) analog-treated seed relative to germinating seed (Li et al., 2005
Brassinosteroids (BRs) are powerful plant hormones involved in vascular differentiation, seed germination, and vegetative growth (Sasse, 2003
The use of Arabidopsis mutants that are insensitive to BR resulted in the identification of several components of the BR signaling pathway. The BR receptor BR-INSENSITIVE1 (BRI1) encodes a plasma membrane localized Leu-rich repeat kinase with an extracellular domain that binds brassinolide (BL), the most physiologically active BR. Binding of BL to BRI1 results in phosphorylation of the kinase domain that activates the BRI1 protein leading to BR responses (Friedrichsen et al., 2000
Loss-of-function genetic screens have identified additional BR signaling components downstream of BRI1 and BAK1. These include bri2, a negative regulator of the BR pathway, which displays bri1-like phenotypes including dwarfism and BR-insensitive and ABA-hypersensitive responses (Li et al., 2001
Evidence has accumulated of cross talk between BRs and other hormones including auxin, ABA, jasmonic acid, and ethylene (Krishna, 2003 In this study, we report on an HSD-like gene from Arabidopsis referred to as AtHSD1. Plants overexpressing AtHSD1 constitutively expressed BR response genes and displayed similar phenotypes to those overproducing BRs or BRI1.
Structural Analysis of AtHSD1
We studied an Arabidopsis gene that is annotated by the Munich Information Centre for Protein Sequences Arabidopsis Genome Database as a homolog of human and animal 11
Phenotypes Resulting from Constitutive Overexpression of AtHSD1 Are Similar to Those Produced by Overproducing BRs or BRI Genes The AtHSD1 cDNA was fused to the cauliflower mosaic virus 35S promoter to drive high levels of gene expression in vegetative tissues. After transformation and selection, 42 kanamycin-resistant T1 transgenic plants were obtained. Reverse transcription (RT)-PCR analysis confirmed higher AtHSD1 mRNA level in T3 plants of four transgenic lines overexpressing AtHSD (AOHSD lines) relative to wild type (Fig. 2A ).
The effects of AtHSD1 overexpression on growth of AOHSD plants were monitored during plant development. The number of rosette leaves, flowering time, and flower morphology were not significantly different between wild-type and transgenic plants. However, as shown in Figure 2, B and C, the AOHSD lines outgrew the wild type and mature AOHSD plants were reproducibly approximately 20% taller (43.0 ± 1.4 cm) than wild-type plants (35.8 ± 1.0 cm). Stem diameter was consistently larger in transgenics than in wild type, and the number of branches and siliques were also greater in transgenics than in wild-type plants (Fig. 2C), leading to a doubling of seed weight per plant in two transgenic lines relative to wild type (Fig. 2F). The higher seed yield was due to an increased number of siliques per plant and seed size was not significantly increased (data not shown). The roots of AOHSD plants grown on Murashige and Skoog medium for 7 d were 23% longer (4.8 ± 0.5 cm) than those of wild-type plants (3.9 ± 0.4 cm). Similarly, plants overexpressing AtHSD1 in canola (BOHSD plants) displayed increased growth from the seedling stage to flowering (Fig. 2, D and E). Increased stem diameter was also observed in BOHSD plants relative to wild type. In three transgenic lines (T1 generation), average diameters after 68 d of plant growth (measured just above soil level) were 113%, 127%, and 152% of stems in untransformed controls (average diameter of controls was 0.82 cm).
Overall, these results are similar to the phenotypes produced by overexpression of DWARF4, which encodes a steroid 22
Arabidopsis seeds exhibit primary dormancy (Bentsink and Koornneef, 2002
BRZ blocks BR biosynthesis at the C-22 hydroxylation step (Asami et al., 2000 ) and was used to test whether AtHSD1 effects require BRs. Figure 4
showed that both AOHSD and wild-type seedlings were sensitive to BRZ under both light and dark. Therefore, AtHSD1 effects depend on the presence of BRs and AtHSD1 overexpression appears to increase the sensitivity of plants to BRs.
Loss-of-Function Plants Show Semidwarfed Phenotype and Insensitivity to BL
There were no apparent phenotypes in AtHSD1 T-DNA knockout lines, probably because phenotypic effects were masked by gene redundancy. To produce plants with reduced AtHSD gene expression, an AtHSD1 RNA interference (RNAi) transformation was conducted. Six out of 15 transgenic lines displayed a semidwarf phenotype with slightly darker green, wider leaves, and shorter petioles than wild-type plants and typical examples are shown in Figure 5, A and B
. Semiquantatitive PCR showed AtHSD1 expression was substantially reduced in a representative RNAi plant relative to wild type (Fig. 5C), and transcripts of other gene family members were also reduced in RNAi plants (data not shown). The phenotypes produced by the RNAi lines are similar to those exhibited by bes1, which contains a defective transcription factor mediating BR-regulated gene expressions (Yin et al., 2005
AtHSD Gene Expression Affects Sensitivity to ABA and ABA Metabolism
ABA and BRs have been shown to act antagonistically (Mandava, 1988
To investigate the relationship between ABA metabolism and the ABA insensitivity of AOHSD lines, we profiled ABA and its metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry (Feurtado et al., 2004 ). There are several pathways of ABA catabolism involving either hydroxylation or conjugation. In most cases, the predominant pathway of ABA catabolism is via hydroxylation at the 8' position to yield phaseic acid (PA), which is then reduced to dihydrophaseic acid (Zaharia et al., 2005). As expected (Fig. 6B), the ABA content of freshly harvested wild-type seeds (292 ng/g dry weight) is more than that of seeds stored 4 weeks at room temperature (168 ng/g dry weight), consistent with increased germination after storage (Fig. 3C). The ABA contents of both freshly harvested and stored seeds of AOHSD lines (326 and 344 ng/g dry weight, respectively) are a little higher than in wild-type seeds. After 2 d treatment of stored wild-type seeds with 2 and 10 µM (+)-ABA, the tissue ABA levels (1,360 and 6,570 ng/g dry weight, respectively) were much higher than in untreated wild-type seeds (168 ng/g dry weight). In contrast, the ABA levels in AOHSD seeds were lower after ABA treatments (717 ng/g dry weight in the 2 µM treatment and 2,140 ng/g dry weight in the 10 µM ABA treatment) than in wild-type seeds, but the levels of all ABA metabolites were higher than in wild-type seeds (Fig. 6B). For example, PA only significantly accumulated in ABA-treated AOHSD seed. The elevated presence of ABA catabolites and reduced ABA content in AOHSD seed relative to wild type are consistent with increased flux through the ABA metabolic pathway. Wild-type seeds did not germinate after ABA treatment (Fig. 6A) or if they were untreated but freshly harvested (Fig. 3B). However, AOHSD seeds can germinate and grow well despite the presence of high concentrations of applied ABA (Fig. 6A). The results described above suggest that AOHSD seeds have a higher capacity for ABA metabolism that is manifested in greater catabolism of exogenously applied ABA. However, despite increased ABA catabolism in AOHSD seeds, it is noteworthy that ABA nonetheless accumulates to high levels (e.g. to 2,140 ng/g dry weight in AOHSD seeds treated with 10 µM ABA) but the seeds nonetheless germinate readily. Therefore, the transgenic seeds demonstrate markedly reduced sensitivity to ABA.
To determine if AtHSD1 is responsive to BL, the most active BR, a time course of BL treatment was performed. Induction of AtHSD1 occurred rapidly with a maximum around 2 h. Treatment with other plant hormones, such as ABA and GA3, did not significantly induce gene expression (Fig. 7A ). On the other hand, AtHSD1 expression was severely reduced in all BR-deficient mutants tested, such as cbb1, cbb3, and det2 (Fig. 7B), suggesting that AtHSD1 was induced specifically by BRs during plant growth. To further test the genetic relationship between AtHSD1 and BR perception, we treated bri1 seedlings with BL. The results showed that the induction of AtHSD1 was reduced in bri1 (Fig. 7C), suggesting that AtHSD1 transcription is dependent on BR perception.
Differential Expression of Genes in Overexpression Plants
To examine the effects of AtHSD1 gene expression on other genes, expression profiling was conducted using Arabidopsis cDNA microarrays. Comparisons were made between 4-week-old seedlings of wild-type (Col) and AOHSD lines (Table I
) and the experiments were repeated four times. Statistical analysis using Significant Analysis of Microarrays (SAM) showed 127 genes to be significantly differentially expressed by more than 2-fold, including 38 up-regulated genes and 89 down-regulated. Among the 38 induced genes, there were several with functions similar to those of known BL-induced genes encoding putative cell elongation or expansion-associated proteins such as pectinesterase and xyloglucan fucosyltransferase. BRs are known to increase expression of many genes involved in cell wall biosynthesis and modification, consistent with their effects on increasing cell expansion and division (Haubrick and Assmann, 2006
The BR ENHANCED EXPRESSION3 (BEE3) gene encodes a basic helix-loop-helix transcription factor that is induced by BR and is a positive regulator of BR responses. The bri1 SUPPRESSOR gene (BRS) encodes a secreted Ser carboxypeptidase that acts as a negative regulator of BR action, although its true mode of action is unknown (Haubrick and Assmann, 2006 ). Expression of both of these genes is significantly reduced in genotypes with defects in BR synthesis or responses (Fig. 8
). However, expression of these genes is slightly increased in AOHSD plants and this result, together with the increased expression of BR-related genes in Table I, suggests elevated BR signaling and responses in plants overexpressing HSD.
AtHSD1 Expression Is Tissue Specific
An AtHSD1 promoter-reporter gene fusion was employed to study the tissue specificity of AtHSD1 expression. The promoter region of AtHSD1 (approximately 1.5 kb) was isolated from Arabidopsis genomic DNA and fused to the GUS protein coding sequence (PHSD1:GUS). Histochemical analysis of GUS activity in transgenic plants harboring PHSD1:GUS showed a high level of expression in the above-ground parts of seedlings, and weak expression in root tissues in both light and darkness (Figs. 9, A–D
and 8H). PHSD1:GUS was strongly expressed in vascular tissues (Fig. 9E) and this is consistent with the known involvement of BRs in vascular differentiation (Sasse, 2003
Increased Stress Tolerance of AOHSD and BOHSD Plants The abiotic stress tolerance of plants overexpressing P35S:HSD were assessed by their ability to tolerate a saline growth medium. Wild-type Arabidopsis plants died after application of 300 mM NaCl, whereas transgenic plants were healthy and unbleached at 400 mM (Fig. 10A ), although their rate of growth was reduced relative to untreated material. The effect of salt stress on seedling vigor was also assessed. Both wild-type and transgenic seeds of both Arabidopsis and canola were germinated on medium containing 100 mM NaCl and after 2 weeks a significant number of wild-type seedlings had died, whereas only a few of the transgenic seedlings failed to grow. Furthermore, wild-type seedlings were stunted and discolored, whereas transgenic seeds appeared healthy (Fig. 10B).
We have characterized the functional effects of a gene with homology to animal 11 -HSD. Although 11-hydroxysteroids are important in regulating growth and development in animals, they have not been found in plants so the possible enzymatic functions of plant HSDs are uncertain. Enzyme assays of AtHSD have demonstrated that it possesses 11- and 17-HSD activity (d'Andrea et al., 2007) but its true substrate(s) is/are unknown. Our data suggest that AtHSD produces BR-like effects such as increased growth, branching, and flower production as well as thicker stems and increased stress tolerance. In addition, expression of the native HSD gene was induced by BL. Although GAs are also associated with rapid growth and increased height (e.g. Coles et al., 1999), we observed that exogenous application of GA produced a more elongated type of growth than that produced in AOHSD and BOHSD plants or by application of BL to control plants. Furthermore, treatment of plants with GAs or transgenic overproduction of GAs has not, to our knowledge, been associated with increased stress tolerance. On the other hand, the increased ABA catabolism and reduced sensitivity to ABA that we measured in AOHSD plants cannot account for the increased growth observed in OHSD plants and for their enhanced stress tolerance. Typically, ABA-deficient and ABA-insensitive mutants grow no better than wild type and are susceptible to water loss (e.g. Leon-Kloosterziel et al., 1996). Conversely, although ABA-hypersensitive mutants are more tolerant of drought stress, they typically grow slowly (e.g. Pei et al., 1998). However, the role of HSD in relation to BR action has not been established. One possibility is that AtHSD is responsible for catalyzing a step in the biosynthesis of these hormones. However, the possibility that AtHSD functions in BR signaling cannot be excluded. For example, the fact that OHSD plants are hypersensitive to BR and that hsd (RNAi) plants are relatively insensitive to BR (Fig. 5) is more consistent with a role in mediating responses to BR than in controlling levels of BR.
Previous reports have suggested a reciprocal relationship between BR and ABA. The experiments reported here also provide examples of this phenomenon. AOHSD plants are hypersensitive to BR and insensitive to ABA and conversely, RNAi plants are insensitive to BR. We also show that enhanced BR effects in AOHSD seeds are associated with a greater ability to catabolize exogenous ABA, suggesting either faster ABA metabolic flux or at least a higher capacity for ABA metabolism. The antagonistic relationship between ABA and BR raises the puzzling question of how BR-like effects of HSD can be associated with enhanced stress tolerance (which is promoted by ABA) as observed in this study (Fig. 9) and in previous ones (Krishna, 2003 The interaction between ABA and BR is clearly complex: partially antagonistic and partially synergistic. A full understanding of the role that HSD plays in this hormonal interaction will require further experiments to define its mode of action. For example, analysis of the effects of HSD expression on active BR levels will be required to characterize the relationship of HSD to BR biosynthesis. Genetic experiments to determine whether HSD overexpression complements BR-deficient and/or BR-insensitive mutants are also required. However, irrespective of its precise mode of action, AtHSD is clearly an important contributor to plant growth and development and provides the potential for producing increased yield and stress tolerance in crop plants.
Plant Materials
Arabidopsis (Arabidopsis thaliana) ecotype Col-0 was used as the wild type, and Arabidopsis transgenic plants were transformed with Agrobacterium tumefaciens GV3101 using the floral dip method (Clough and Bent, 1998 The cbb1 and cbb3 mutant seeds were kindly provided by Dr. Carsten Müssig, and the det2 mutant seeds by Dr. Jianming Li. bri1 (cbb2, CS292) mutant seeds were obtained from the Arabidopsis Biological Resource Center. For the mRNA analysis following various hormone treatments, 4-week-old wild-type or mutant plants were sprayed with 1 µM BL (OlChemIm Ltd., http://www.olchemim.cz/INDEX_e.HTM), 100 µM (+)-ABA, or 5 µM GA (GA4+7), respectively, and incubated for varying times. For (+)-ABA dose response curves, seeds (>120) were germinated and grown in light on Murashige and Skoog medium containing varying concentrations of (+)-ABA and germination was scored after 7 d. The seeds were treated for 2 d on Murashige and Skoog medium containing 2 and 10 µM (+)-ABA, respectively, then collected, washed, and frozen in liquid nitrogen.
A genome database search resulted in the identification of a genomic sequence encoding a protein that shares high homology with human and animal HSD, especially in the N-terminal region. We named this gene AtHSD1. The coding region of AtHSD1 gene was generated by PCR amplification of plasmid Uvi51 containing full coding sequence of this gene, the primers 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGAGTTGATAAACGACTTTCTC-3' and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATCCGACTTGATTTCTGGAGT-3' (attB sites for recombination cloning are shown in bold, and the sequence corresponding to AtHSD1 is underlined) were used for PCR. For generating AtHSD1 overexpression lines, the PCR product was introduced into the binary vector pK7WG2 (Karimi et al., 2002
The region of AtHSD1 from –1,534 to +75 bp was generated by PCR amplification of genomic DNA using primers 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCAATGGAACCGAAAGCCTAA-3' and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGGCAAGAAGAAGCAGAGACC-3' (attB sites for recombination cloning are shown in bold, and the sequence corresponding to AtHSD1 is underlined). The resultant 1,609-bp fragment was subcloned into binary vector pMDC163 (Curtis and Grossniklaus, 2003 For GUS staining, seedlings were immersed in 50 mM sodium phosphate buffer (pH 7.2) with 1 mM 5-bromo-4-chloro-3indolyl-D-GlcUA and incubated at 37°C for 12 h. Chlorophyll was extracted by passing through increasing concentrations of ethanol. Nineteen independent T2 transgenic lines were analyzed and commonly observed staining patterns were recorded.
Total RNA (2.5 µg) from each sample, treated with RNase-free DNase (Promega), was used for reverse transcriptase reactions. First-strand cDNA was synthesized with random hexamers using a SuperScript first-strand synthesis system according to the manufacturer's instructions (Invitrogen Life Technologies), and QuantumRNA 18S internal standards (Ambion) were used as a positive control for quantification of the relative amounts of cDNA. One microliter of RT reaction mixture was used as a template in a 20 µL PCR. For AtHSD amplification, the primers HSD1-1 (5'-TGCCAAGTCGATAGTGAACG-3') and -2 (5'-CAGTAACCGACAACCCCACT-3') were used for PCR. For amplification of BRS1, the primers BRS1-1 (5'-CCAACCAAAAGTGGCATTCT-3') and BRS-2 (5'-TGTTGATACGAAACGGTCCA-3') were employed. For amplification of BEE3, the primers BEE3-1 (5'-CGACGAGGGAAAATAAACGA-3') and BEE3-2 (5'-CATGGATTCCACAGCATCAG-3') were employed. The amplification conditions were 94°C (30 s), 56°C (30 s), and 72°C (30 s) and 28 cycles. RT-PCR was repeated twice.
HPLC was performed using a Waters 2695 separation module (Waters). The extraction and purification of ABA and its metabolites, HPLC conditions, addition of internal standards, mass spectrometry, and quantification of endogenous levels of compounds were performed as described by Feurtado et al. (2004)
Four-week-old seedlings of AOHSD and wild-type plants were harvested and frozen quickly in liquid nitrogen. Total RNA was extracted using RNeasy mini kits (Qiagen). Each total RNA sample (50 µg) was converted to cDNA and labeled using the Cyscribe Post-Labeling kit (Amersham Bioscience, RPN5660) following the manufacturer's instructions. The CyScribe GFX Purification kit (Amersham Bioscience, RPN5660X) was used to purify the fluorescently labeled cDNA probe by removing free nucleotides and unincorporated CyDye molecules. Arabidopsis 12 K cDNA microarrays (average length approximately 300 bases) from the Keck Foundation Biotechnology Resource Laboratory at Yale University School of Medicine were used. Hybridized slides were scanned sequentially for Cy3- and Cy5-labeled probes with a ScanArray 4000 laser scanner at a resolution of 10 Am. The experiments were repeated four times. Image analysis and signal quantification were performed using Quantarray (GSI Lumonics). Clones showing a signal value <800 in both Cy3 and Cy5 channels were eliminated from the analysis. The average of the resulting total Cy5 and Cy3 signals were used to calculate the ratios that were used for normalization. Data storage, preliminary data processing, and Lowess normalization were performed with the Bioarray Software Environment (Saal et al., 2002
The salt sensitivity of plants was evaluated by growth in pots in a controlled environment chamber. After 2 weeks of growth under normal conditions, plants were flooded once a week with solutions containing varying NaCl concentrations.
We thank Andrew Ross and Steve Ambrose for liquid chromatography-mass spectrometry analyses, Li Forseille for assistance with some of the canola experiments, and Suzanne Abrams for helpful discussions. In addition, we thank one of the anonymous reviewers for drawing our attention to some relevant publications. Received April 3, 2007; accepted June 22, 2007; published July 6, 2007.
1 This work was supported by the Genome and Health initiative of the National Research Council of Canada and by Genome Canada and Genome Prairie under the program "Enhancing canola through genomics." This article is National Research Council of Canada number 48416. 
2 Present address: Department of Botany, 6270 University Blvd., University of British Columbia, Vancouver, BC, Canada V6T 1Z4. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Adrian J. Cutler (adrian.cutler{at}nrc-cnrc.gc.ca). www.plantphysiol.org/cgi/doi/10.1104/pp.107.100560 * Corresponding author; e-mail adrian.cutler{at}nrc-cnrc.gc.ca.
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ABSTRACT
RESULTS
hydroxylase involved in BR biosynthesis (Choe et al., 2001
), AOHSD (
), and hsd (RNAi; 
) seeds. Percent germination was determined from a minimum of 120 seeds. B, Endogenous ABA and its metabolites in AOHSD and wild-type seeds under different conditions. Fresh, Freshly isolated seed; Stored, seed stored for 4 weeks at room temperature; 2-, stored seed treated with 2 µM (+)-ABA for 2 d; 10-, stored seed treated with 10 µM (+)-ABA for 2 d; ABA-GE, ABA-Glc ester; DPA, dihydrophaseic acid; 7'-OH-ABA, 7'-hydroxy ABA; n-PA, neo-PA.
0.005; Tusher et al., 2001