INTRODUCTION

Brassinosteroids (BRs) are natural plant growth-promoting products, which have structures similar to animal steroid hormones and are distributed throughout the plant kingdom 1. Exogenous application of BR at nanomolar to micromolar levels causes a number of physiological responses. BR-deficient and -insensitive mutants exhibit severe alterations in plant growth and development, such as de-etiolation, dramatic dwarfism, reduced fertility, and extended longevity 2. In addition to a role in plant growth and development, BR has been also implicated in the modulation of plant stress responses, including enhancement of chilling-, thermo-, salt-tolerance and protection of plant from the mild drought injury and pathogen attack 2. Till now, at least eight genes have been confirmed to lead to the BR biosynthetic deficiency 3. The characterization of these mutants leads not only to the confirmation of its essential role in plant growth and development as a new class of plant hormone, but also to the elucidation of BR biosynthetic pathway 4.

The first invaluable insight regarding the BR signal transduction comes from the identification and characterization of a BR-insensitive mutant, bri1 5, 6. BRI1 is a membrane-located receptor kinase and recent works have confirmed its critical role in perceiving and transducing BR signal as a membrane-located receptor 7, 8. Great advances have been achieved recently by the identification and characterization of other signal molecules involved in this pathway, including BSR1, BIN2, and BAK19, 10, 11, 12, 13. On the other hand, some works focus on the BR-regulated genes in attempting to elucidate the molecular mechanism of BR-mediated development. The early observations that BR regulates the expression of BRU1 in soybean 14 and TCH4 in Arabidopsis 15 provide molecular evidences that BR is involved in regulation of cell elongation and expansion. Our primary result that BR induces CycD3 transcription in Arabidopsis represents the finding of BR-regulated cell division 16. BR also mediates light-dependent development 17 and their signals are integrated via a dark-induced G protein 18. Some other BR-regulated genes and signaling components have been characterized recently, including Cdc2b, KOR and BAS119, 20, 21. Furthermore, a number of BR response genes have been identified using DNA array approach 22, 23, 24. The characterization of these genes will greatly further our understanding of molecular mechanism of BR action and signal transduction.

RAV1 is a putative DNA binding protein with two distinct types of DNA binding domains, AP2 and VP1/B325. AP2 domain was first identified as a DNA binding domain in a family of tobacco ethylene response element binding proteins (EREBPs) 26 and in Arabidopsis APETALA2 (AP2), a transcriptional factor involved in flower development 27. The number of different proteins containing the AP2 domain appears to be quite large in plants 28, 29. Some of them, such as Arabidopsis ANT, TINY and CBF1, have been shown to be involved in flower development,cell proliferation, and plant responses to hormones and stresses 27, 30, 31. VP1/B3 is another DNA binding domain conserved in a number of DNA binding proteins, such as VP1, ABI3 and ARF1, which have been shown to mediate the plant responses to ABA and auxin 32, 33, 34. RAV1 containing both AP2 and VP1/B3 domains suggests that it represents a new group of DNA binding proteins unique to higher plants. However, little is known about its role in plant growth and development 25.

By a cDNA array, we screened for BR response genes in Arabidopsis det2 cell culture and 53 clones were identified to be responsive to epibrassinolide (epiBL) treatment, including CycD3 and RAV1 22. Here we report that the transcription of RAV1 is down-regulated by epiBL treatment and this regulation of RAV1 by epiBL seems not to require the functional BRI1. Overexpression and under-expression of RAV1 in Arabidopsis result in a retardation of lateral root and rosette leaf development and earlier flowering, respectively. Our results suggest that RAV1 may act as a negative growth regulator in a new BR signal pathway during growth and development.

MATERIALS AND METHODS

Suspension culture and plant growth

Seeds of Arabidopsis thaliana BR-deficient mutant det2 35 and BR-insensitive mutant bri1-1 6 were surface sterilized and cultured on B5 medium containing 2% glucose, 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.45 μM kinetin (KT) in darkness at 25°C for callus induction. The suspension cultures were established and propagated under conditions described previously 16.

The wild-type Arabidopsis (Col-0 ecotype) was used for analysis and transformation. Plants were grown basically on vermiculite saturated with 0.3×B5 medium under continuous illumination (80-120 μEm−2sec−2) at 23°C 36.

Plant hormone and inhibitor treatment

The hormone-starved cells were treated with 24-epibrassinolide (epiBL), 2,4-D, Zeatin (ZT) and/or inhibitors as previously described 16. The 30-day-old plants of wild-type Col-0 were sprayed with 1 μM epiBL to examine the response.

RNA and DNA gel blot analysis

Total RNA was isolated with guanadine thiocyanate buffer 37. RNA gel blot analysis was performed according to procedures described previously 16. Probes were prepared with full-length RAV1 cDNA using a random labeling system (Amersham, UK). The anti-RAV1 RNA probe was synthesized by T7 RNA polymerase and used for expression analysis of RAV1 in transgenic plants.

The genomic DNA was prepared from 30-day-old plants by CTAB method 38 and completely digested with appropriate enzymes. The DNA was separated on a 0.8% agarose gel, transferred onto a nylon filter (Hybond N+, Amersham) and probed with RAV1.

Western blot analysis

A Sac I and Dra I fragment of RAV1 cDNA (∼1.1 kb) without start codon was cloned into the expression vector pGEX-3X (Pharmacia) digested with SmaI and introduced into E. coli by electroporation to generate the GST-RAV1 fusion protein. The expression of fusion protein, antibody preparation and immunological detection were conducted according to the methods described previously 39. Proteins were extracted from det2 cells treated with/without epiBL as described by Fawcett et al 40.

Sense- and antisense-RAV1 plasmid construction and plant transformation

To generate two orientation insertions of RAV1 cDNA, an 11 bp multiple clone sites containing a KpnI site from pUC118 vector was introduced into pBI121 to form pJL700. An XhoI fragment of full-length RAV1 cDNA (1.23 kb) was cloned into pBluescript II SK (+) to generate two plasmids with both trans- and reverse-orientated insertions. The KpnI and XbaI fragments from both plasmids were ligated to pJL700 to generate sense- and antisense-RAV1 plasmids, pJL781 and pJL782, respectively. Both pJL781 and pJL782 were introduced into Agrobacterium strain GV3101 by electroporation and were used to transform wild-type Col-0 plants by vacuum infiltration 41. The transformants were selected in MS medium containing 50 mgL−1 kanamycin. The transgenic plants were self-pollinated to generate homozygous transgenic lines and confirmed by DNA gel blot analysis with a CaMV 35S promoter probe. Two independent T4 homozygous lines of each construct were used for molecular and phenotypic characterization.

For morphological observation of seedlings, seeds of RAV1 transgenic lines and wild-type Col-0 were germinated in MS plates containing 2% sucrose and grown vertically in a chamber at 23 °C with 16 h light/ 8 h dark photoperiod. The lateral root number was counted under microscope.

RESULTS

RAV1 is down-regulated by epibrassinolide

To identify BR response genes (BRR), we used cDNA array to monitor gene expression of Arabidopsis det2 suspension cultures treated with 24-epibrassinolide (epiBL). In a total of 13,000 arrayed cDNA clones, 53 (desig-nated as BRR1-BRR53) were found to be BR responsive 22. Sequencing and homology analyses showed that the BRR8, a BR down-regulated clone with a 1.23 kb cDNA, is identical to RAV1 gene (EMBL GenBank accession No. AB013886, At1g13260).

To confirm and further understand the regulation of RAV1 by BR, RNA gel blot analyses of det2 cells and wild type Col-0 seedlings were performed. When det2 cells were treated with different concentrations of epiBL, RAV1 mRNA decreased in a dose-dependent manner and over 1 μM epiBL effectively repressed RAV1 transcription (Fig 1A). Kinetics study showed that the most dramatic repression occurred when det2 cells were incubated for 4 h with 5 μM epiBL (Fig 1B). We further investigated this down-regulation in the wild-type plant. As shown in Fig 1C, 30-day-old seedlings of wild-type plants showed an apparent decrease in RAV1 mRNA level at 24 h after treatment of 1 μM epiBL. Western blot analysis in the det2 cells showed that incubation with 5 μM epiBL for 4 h caused about 50% decrease of RAV1 level (Fig 1D). These results indicate that RAV1 is down-regulated by BR.

Figure 1
figure 1

Down-regulation of RAV1 by epibrassinolide. (A-C)The transcriptional repression of RAV1 by epibrassinolide (epiBL). All data shown were calibrated against the RNA loadings. (A) The det2 cells were treated with different concentrations of epiBL or DMSO (CK) for 4 h. (B) The det2 cells were incubated in the medium supplemented with 5 μM epiBL for various times after hormone starvation. (C) 30-day-old plants of wild-type Col-0 were sprayed with 1 μM epiBL or equal concentration of DMSO (CK) and harvested after 24 h. (D) Western blot analysis of RAV1 in the epiBL-treated det2 cells. Proteins were isolated from det2 cells treated with 5 μM epiBL or equal volume of DMSO (CK) for 4 h and RAV1 was determined by the affinity with purified anti-GST-RAV1 antibody. The lower non-specific bind served as the loading control.

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Responses of RAV1 to other hormones essential for plant growth and development were also examined. As shown in Fig 2, no obvious alteration of RAV1 expression was found in cells treated with 2,4-D. However, a slight repression of RAV1 transcription by zeatin was observed and zeatin showed some synergistic effect with epiBL. These results indicate that RAV1 is mainly responsive to BR, and also to a less extent to cytokinin.

Figure 2
figure 2

The effect of auxin and cytokinin on RAV1 expression. The RNA was isolated from det2 cells treated for 4 h with 2,4-D (4.5 μM), ZT (1 μM), epiBL (5 μM) and their combinations indicated or equal volume of DMSO (CK).

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RAV1 is a single copy gene

The putative amino acid sequence of RAV1 contains two different DNA binding domains, an AP2 near N-terminal and a B3 in C-terminal 25. Blast analysis showed that in the Arabidopsis genome there are 5 other putative genes that encode both B3 and AP2 domains (At3g25730, At1g68840, At1g25560, At1g50680 and At1g51120) with 41.4-67.7% identity to RAV1, suggesting that RAV1 belongs to a small novel gene family. However, DNA gel blot analysis under high stringency with full-length RAV1 cDNA probe demonstrated that RAV1 is a single copy gene in Arabidopsis genome (Fig 3).

Figure 3
figure 3

DNA gel blot analysis of RAV1 gene in the Arabidopsis genome. Wild-type Col-0 genomic DNA was digested with the enzymes indicated and transferred onto a nylon filter. The filter was probed with RAV1 fragment and the probed DNA marker was shown at left.

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Transcriptional regulation of RAV1 by epi-brassin-olide does not require the function of BRI1

To investigate the pathway leading to down-regulation of RAV1 transcription by BR, we examined RAV1 expression in det2 cells treated with inhibitors widely used in signal transduction research. Okadaic acid (OA), a phosphatase inhibitor, showed no effects on RAV1 repression by epiBL, nor did the strauroprine (St), a broad range inhibitor of protein kinase (Fig 4A), suggesting that protein phosphorylation or dephosphorylation is unlikely involved in the pathway leading to BR-regulated RAV1 transcription. Interestingly, the presence of protein synthesis inhibitor cyclohexmide (Chx) dramatically induced RAV1 expression (Fig 4B), suggesting that a short-lived repressor is involved in RAV1 transcriptional control. However, the repression by epiBL was also observed even in the presence of Chx (Fig 4B), implicating that the newly synthesized protein is not essential for BR-regulated RAV1 transcription.

Figure 4
figure 4

Pathway analysis of epiBL-regulated RAV1 transcription. (A) Protein phosphatase inhibitor okadaic acid (OA) and kinase inhibitor staurosporine (St) showed no effect on the repression by epiBL. The RNA was extracted from det2 cells incubated for 4 h with DMSO (CK), epiBL (5 mM), OA (0.1 mM), St (1 mM) and their combinations as indicated. (B) Protein synthesis inhibitor cycloheximide (Chx) greatly induced RAV1 but showed no obvious effect on the down-regulation by epiBL. The RNA was prepared from det2 cells pretreated with ethanol (CK and epiBL) or 100 mM Chx for 1 h and then for additional 4 h with 5 mM epiBL (Chx+epiBL) or with equal volume DMSO (CK and Chx). (C) Transcritional down-regulation of RAV1 by epiBL in bri1-1 mutant cells. The RNA was from bri1 cells treated with 5 mM epiBL for 0, 1, 2 and 4 h after hormone starvation.

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Similar to our previous observation in CycD3 induction by epiBL 16, it seems that BRI1 is not essential for BR-regulated RAV1 expression. To test this hypothesis, an RNA gel blot analysis was carried out in the suspension culture of bri1-1, a BR-insensitive mutant in which BRI1 pathway is blocked 6. When bri1-1 cells were treated with 5 μM epiBL, RAV1 transcripts decreased considerably (Fig 4C) to a level even lower than that in det2 cells (Fig 1B). These results imply that regulation of RAV1 by BR does not require functional BRI1.

Alteration of RAV1 expression affects lateral root and rosette leaf development and flowering time

To understand the role of RAV1 in plants, sense and antisense RNA constructs with full-length RAV1 cDNA (1.23 kb) driven by cauliflower mosaic virus 35S promoter were introduced into wild-type plants by Agroba-cterium-mediated transformation. 112 and 78 T1 plants of overexpressing (RAV1-O) and underexpressing (RAV1-U) RAV1 were obtained by kanamycin selection, respectively. After co-segregation and DNA gel blot analysis, two independent T4 homozygous lines in each construct were selected for further characterization. RNA gel blot analysis with anti-RAV1 probe revealed that compared to wild-type, 20-day-old seedlings of RAV1-U lines showed a relatively lower RAV1 expression, but two RAV1-O lines had much higher levels of RAV1 transcripts (Fig 5A). Surprisingly, an obvious RNA degradation was observed in these two RAV1-O lines (Fig 5A). We then investigated some of other RAV1-O lines and found that the RNA degradation occurred in almost all examined lines (data not shown).

Figure 5
figure 5

Molecular and morphological characterization of RAV1 transgenic plants. (A) RAV1 expression analysis with 20-day-old plants of wild-type Col-0 (WT), 2 independent RAV1 overexpression (RAV1-O) and underexpression (RAV1-U) lines. Blot was probed with the anti-RAV1 RNA probe. (B) The phenotype of lateral roots and rosettes of WT and RAV1 transgenic seedlings. Seedlings were grown vertically on MS medium at 23°C for14 days. (C) The 30 day-old plants of wild-type and RAV1 transgenic plants.

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Although RAV1-O and RAV1-U transgenic plants were almost indistinguishable from the wild type after flowering (data not shown), differences could be observed at the early developmental stage. When grown vertically on MS plates, seedlings of RAV1-O lines had apparently less lateral roots and rosette leaves compared to those of wild-type and RAV1-U lines, with decreases of about 30% and 15%, respectively (Tab 1 and Fig 5B). However, no obvious differences of lateral root and rosette leaf number were found between the wild-type and RAV1-U plants (Tab 1), though the length of lateral roots of RAV1-U seedlings was somewhat shorter than that of the wild-type plants (Fig 5B). These results indicate that overexpression of RAV1 has a retardatory effect on development of the lateral root and rosette leaf.

Table 1 Lateral root, rosette leaf number and flowering time of wild-type and RAV1 transgenic plants
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Grown in plates, RAV1-U seedlings exhibited an earlier inflorescence initiation compared to the wild-type and RAV1-O plants. Further examination of these plants in greenhouse indicated that RAV-U plants flowered 4.8 days and 6.6 days earlier than the wild-type and RAV1-O plants, respectively (Tab 1 and Fig 5C). However, the flowering time of RAV1-O plants was delayed 1.8 days compared to that of the wild-type plants, suggesting that underex-pression of RAV1 appears to accelerates the development of Arabidopsis seedlings.

DISCUSSION

RAV1 is a primary BR response gene

By cDNA array and RNA gel blot analysis, we found that RAV1 is down-regulated by epiBL, indicating that RAV1 is a BR response gene. RAV1 mRNA is ubiquitously presented in all Arabidopsis organs, including roots, rosette leaves, cauline leaves, inflorescent stems, flowers, and siliques. The RAV1 expression is relatively high in roots, leaves and stems, but very low in flowers 25. Our finding that RAV1 is a BR down-regulated gene may partly account for the different expression levels in various organs. For example, a high level of BR in flower is accompanied by a very low level of RAV1 transcripts, and a low level of BR in roots, stems and leaves by a higher expression of RAV1. Therefore, the RAV1 expression and BR distribution in plants are consistent with the finding that RAV1 is a BR down-regulated gene.

Protein synthesis inhibitor Chx can greatly induce the transcription of most early auxin response genes, AUX/IAA 42. The dramatic induction of RAV1 by Chx suggests that there is a repressor involved in RAV1 transcriptional regulation. However, the observation of down-regulation by epiBL under Chx condition implicates that the newly synthesized protein may not be essential for BR-regulated RAV1 expression or the protein required may have already existed in plant cells. Furthermore, the regulation of RAV1 by epiBL occurrs within 1 h and the protein phosphorylation or dephosphorylation is not involved in this process. All these data demonstrate that RAV1 is a BR primary response gene.

RAV1 may function as a negative growth regulatory component

In plant, B3/VP1 domain was mainly found in some ABA and auxin response factors 32, 34 and a large number of AP2-containing proteins were identified to be the regulatory factors in various developmental aspects and responses 28, 29. The finding that RAV1 contains two unrelated DNA binding domains that bind to two bipartite unrelated sequence motifs separated by various spacing in two different orientations 25 suggests that RAV1 may play a regulatory role in some developmental and/or responsive process in plants. The down-regulation of RAV1 by epiBL indicates that RAV1 is involved in BR-regulated deve-lopment. The observations that the overexpression of RAV1 leads to the retarded lateral root and rosette leaf development and the underexpression causes early flowering, along with the finding of the transcriptional repression by cytokinin and RNA degradation in RAV1 overexpressed plants, suggest that RAV1 functions as a negative growth regulatory factor.

In contrast to some morphological alterations before flowering, the RAV1 transgenic plants were almost indistinguishable from the wild-type in later development stage. This might be partly due to the RNA degradation occurred in RAV1 overexpression lines and may explain why Kagaya et al failed to find apparently morphological changes in overexpressed RAV1 plants 25. According to our results, it is likely that RAV1 only affects the rate of development. Furthermore, the presence of two types of important DNA binding domains confers the possibility that RAV1 might also be involved in some other unidentified responses in plants. Therefore, further work is needed in searching of RAV1 target genes to understand the regulatory mechanism.

BR may regulate RAV1 through a BRI1-independent signal pathway

Our observation that the epiBL-regulated RAV1 expression involves neither protein phosphorylation/dephosphorylation nor protein synthesis and occurs in the bri1 mutant cells suggests that BR regulates RAV1 transcription through a pathway other than the BRI1, a pathway identified and well-characterized so far to perceive and transduce BR signals 3. Our previous analysis of BR-induced CycD3 expression has suggested that apart from BRI1, there may exist an unidentified BR signal pathway through which BR induces CycD3 transcription and promotes cell division 16. Recently, an antisense inhibition of BRI1 receptor in rice also revealed that an additional protein kinase signaling component may function downstream to the perception of BR 43.

In animals, there coexist two pathways to transduce steroid hormone signals. The first involves a membrane-located receptor with an extracellular ligand domain to perceive hormone signals, and an intracellular domain responsible for transducing signal through a protein phosphorylation cascade to mediate some responses 44. The second pathway involves an intracellular steroid-activated receptor complex to directly regulate the transcription of genes by binding to the promoter 45. In plants, although a chaperon heterocomplex similar to that of intracellular steroid receptor in animal has been identified 46, 47, 48, neither the candidate gene encoding a putative intracellular receptor of BR has been found in the Arabidopsis genome 49, nor evidence suggested the existence of BR intracellular receptor. However, three BRI1 homologue were identified in Arabidopsis genome 49. Therefore, it is possible that BR-regulated transcription of CycD3 and RAV1 is through another BRI1 homologues or plant might have an unrelated class of steroid receptors whose identity has not been discovered yet (Fig 6) 50.

Figure 6
figure 6

A model for BR signal transduction. BRI1, a membrane-located BR receptor, interacts with BAK1 and perceives BR signal and transduces it through a cascade of protein phosphorylation and dephosphorylation, causes the non-genomic effect and transcriptional regulation of some BR response genes such as TCH4, Cdc2b and KOR, and then affects cell elongation and some responses. Paralle to BRI1, BR might interact with an unknown receptor or component to regulate expression of some other BR response genes such as CycD3 and RAV1, resulting in the promotion of cell division and other subsequent physiological responses in plants.

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