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First published online February 23, 2007; 10.1104/pp.106.093096 Plant Physiology 143:1680-1688 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Roles of Brassinosteroids and Related mRNAs in Pea Seed Growth and Germination1,[W],[OA]Department of Biosciences, Teikyo University, Utsunomiya 320–8551, Japan (T.N., Y.Y., T.Y.); Center for Research on Wild Plants, Utsunomiya University, Utsunomiya 320–8505, Japan (M.U., Y.T.); and Department of Chemistry, Joetsu University of Education, Joetsu 943–8512, Japan (S.T.)
The levels of endogenous brassinosteroids (BRs) and the expression of the biosynthesis/metabolism/perception genes involved have been investigated during the development and germination of pea (Pisum sativum) seeds. When seeds were rapidly growing, the level of biologically active BRs (brassinolide [BL] and castasterone [CS]) and the transcript levels of two BR C-6 oxidases (CYP85A1 and CYP85A6) reached a maximum, suggesting the significance of BL and CS in seed development. In the early stages of germination, CS, but not BL, appeared and its level increased in the growing tissues in which the transcript level of CYP85A1 and CYP85A6 was high, suggesting the significance of CS in seed germination and early seedling growth of pea. 6-Deoxocathasterone (6-deoxoCT) was the quantitatively major BR in mature seeds. At the early stage of germination, the level of 6-deoxoCT was specifically decreased, whereas the levels of downstream intermediates were increased. It seems that 6-deoxoCT is the major storage BR and is utilized during germination and early growth stages. The level of the mRNAs of BR biosynthesis and perception genes fluctuated during seed development. In mature seeds, most of mRNAs were present, but the level was generally lower compared with immature seeds. However, CYP90A9 mRNA rapidly increased during seed development and reached the maximum in mature seeds. The mRNAs stored in mature pea seeds seem to be utilized when seeds germinate. However, it was found that de novo transcription of mRNAs also starts as early as during seed imbibition.
Generally, immature seeds of plants contain high levels of plant hormones, GAs, cytokinins (CKs), auxins, abscisic acid (ABA), and brassinosteroids (BRs). A number of plants contain both GAs and auxins at the highest levels during early to mid-embryo development, when CKs are decreasing rapidly and there is little or no ABA detectable (Rock and Quatrano, 1995  ). The increases in GAs and auxins in pea (Pisum sativum) and wheat (Triticum aestivum) are correlated with increases in pod and grain length. It thus seems that CKs have physiological roles in the early stage of embryo development, whereas GAs and auxins elicit their activity in the later stages. The level of ABA reaches the highest levels at the middle and late periods of seed development prior to returning to lower levels in the dry seed, resulting in acceleration of a buildup of nutrient reserves, an arrest of tissue growth, development of desiccation tolerance, and prevention of precocious germination (Rock and Quatrano, 1995). Choe et al. (2000) found that the dwarf5 (dwf5) mutant of Arabidopsis (Arabidopsis thaliana), which is BR deficient due to a defect in the sterol  7 reductase, produces aberrantly shaped seeds. The lk mutant of pea, which is severely BR deficient due to the impaired sterol 5 -reductase, also produces irregularly shaped seeds (Fig. 1; Nomura et al., 2004). A BR-deficient Vicia faba bean also produces small seeds (Fukuta et al., 2005). These facts indicate that sterols or BRs may be required for normal seed development.
Germination of seeds is known to be retarded by ABA (Merlot and Giraudat, 1997 ) and, in lettuce (Lactuca sativa), accelerated by the de novo synthesis of GA triggered by red light irradiation (Toyomasu et al., 1998). The synthesis of -amylase associated with seed germination is also accelerated by GAs (Richards et al., 2001). It has been known that germination of aged rice (Oryza sativa) seeds (Yamaguchi et al., 1987) as well as clover broomrape (Orobanche minor Sm.) seeds (Takeuchi et al., 1997) is stimulated by BR. Steber and McCourt (2001) showed that BR rescues the germination phenotype of severe GA biosynthetic mutants and of a GA-insensitive mutant by overcoming ABA-induced inhibition of germination. Furthermore, the germination rate of the dwf5 mutant is lower than that of the wild-type plants (Choe et al., 2000). These observations raise the possibility that BR is needed for normal germination.
However, little knowledge is available that correlates endogenous BR levels with seed growth and maturation, as well as with seed germination. To get such information, we quantified endogenous BR levels in pea seeds and seedlings at the various growth and development stages. We also quantified the transcript levels of BR-related genes. To this end we have cloned various pea genes related to the biosynthesis and metabolism of BRs (see Fig. 2). Previously, some of those genes had been isolated as mutated genes in the lka, lkb, and lk mutants of pea by us. The LKB gene encodes sterol C-24 reductase (Nomura et al., 1997
Identification of Endogenous BRs and Their Quantitative Changes in Developing Pea Seeds Immature to mature seeds of pea were harvested at six growth stages (Fig. 1 ). The levels of endogenous BRs were examined at five of these stages except stage 5, fully expanded "yellowed" seeds, because it has been difficult to harvest a large amount of seed materials exactly in stage 5. From these seeds, two biologically active BRs, castasterone (CS) and brassinolide (BL), were identified together with 6-deoxoBRs belonging to the late C-6 oxidation. 6-Oxo intermediate BRs belonging to the early C-6 oxidation pathway were not detected at any growth stage (see Fig. 2 ). Quantitative changes of the endogenous BRs in these seeds are shown in Table I . The amounts of BL and CS increased toward stage 3 in parallel to the seed weight increase, but drastically decreased in the fully expanded green seeds (stage 4). Both BRs were not detectable in mature dried seeds (stage 6). The level of 6-deoxocastasterone (6-deoxoCS) rapidly increased and reached the highest level at stage 4, whereas it drastically decreased at stage 6. No such dramatic changes were observed for the levels of the upstream biosynthetic intermediate BRs. However, interestingly, high levels of 6-deoxoBRs were still contained in mature seeds. Among these BRs, 6-deoxocathasterone (6-deoxoCT) is the predominant BR, with the amount accounting for 80% of the total BR content.
Cloning of BR Biosynthesis/Metabolism Genes from Pea Seed
Pea cytochrome P450 genes related to BR biosynthesis and metabolism were cloned by PCR-based approaches using oligonucleotide primers derived from the conserved nucleotide sequences in the expressed sequence tags of legumes Glycine max, Lotus japonicus, and Medicago truncatula, as well as in the known genes of Arabidopsis and tomato. The genes we cloned were CYP85A1 and CYP85A6 as DWARF homologs (Jager et al., 2007
A phylogenetic tree of BR biosynthesis/metabolism-related P450s of pea, Arabidopsis, tomato, and rice was constructed using their amino acid sequences (Fig. 3 ). These analyses suggest that the pea P450 genes cloned will have the same functions as the indicated Arabidopsis, tomato, and rice genes. Recently, it was found that CYP85A1 of Arabidopsis and tomato catalyzes the conversion of 6-deoxoCS to CS, whereas Arabidopsis CYP85A2 and tomato CYP85A3 catalyze the conversion of 6-deoxoCS to BL (Kim et al., 2005 ; Nomura et al., 2005). However, we found that both pea CYP85A1 and CYP85A6 catalyze the conversion of 6-deoxoCS to CS but do not produce a significant amount of BL (Jager et al., 2007). It is interesting that pea has two CPD homologs, CYP90A9 and CYP90A10. It has been proposed that the Arabidopsis CYP90A1 gene encodes BR C-23 hydroxylase because its loss-of-function mutant cpd shows a severe dwarfism that is rescued by exogenous applications of BRs, including 23-hydroxylated precursors (Fig. 2; Szekeres et al., 1996). However, recently, Ohnishi et al. (2006) have suggested that CYP90A1 catalyzes a step different from C-23 hydroxylation but an unknown reaction in BR biosynthesis using in vitro enzyme assay. A putative 2-hydroxylase, DDWF1 (CYP92A6), did not fall into any phylogenetic clan of plant steroid hydroxylases, indicating that DDWF1 could be involved in a reaction other than BR biosynthesis.
Fluctuation of Transcript Levels of BR Biosynthesis/Metabolism/Receptor Genes in Developing Pea Seeds The transcript levels of the genes were examined at immature to mature stages 1 to 6 (Fig. 4; Supplemental Fig. S1). The transcripts of the CYP85A1 and CYP85A6 genes reached the highest level at stage 3, but then decreased in the fully expanded green seeds (stage 4) and vanished by stage 6. Interestingly, the CYP85A1 gene was more highly expressed than the CYP85A6 gene through all stages of growth (Fig. 4 ).
Intriguingly, the patterns of expression of the CYP90A9 and CYP90A10 transcripts were in striking contrast. The CYP90A10 transcript level increased toward stage 4, but thereafter sharply decreased. Its level in immature seed was low. However, the CYP90A9 transcript level was quite low until stage 4, but thereafter sharply increased and accumulated to a high level in mature, dry seed. These results suggest that the CYP90A9 and CYP90A10 genes function at quite different stages of growth. The expression of the CYP90D7 gene, although not high at any stage of seed growth, was the highest at stages 3 and 4. It was not detectable at later stages (5 and 6). The transcript levels of LKB, CYP90B8, CYP92A6, CYP734A11, and LKA were less markedly changed, but as a whole gradually decreased as seeds matured. However, the LK gene transcript gently increased and attained the highest level at stage 6. It is worth noting that mRNAs of LK, LKB, CYP90A9, CYP90A10, CYP92A6, CYP734A11, and LKA were still detectable even in mature, dry seeds of pea, but those of CYP90B8, CYP90D7, CYP85A1 and CYP85A6 were scarce or below the detection levels.
The levels of endogenous BRs fluctuated during seed germination as shown in Table III (Fig. 1). In the 16-h imbibed seed, the levels of endogenous BRs were comparable to those of mature seeds, and neither BL nor CS was detected. In the 1-d-old seedlings, CS appeared and its precursors, 6-deoxoCS, 6-deoxotyphasterol (6-deoxoTY) and 6-deoxo-3-dehydroteasterone (6-deoxo3DT), were markedly increased. In contrast, 6-deoxoCT was decreased to one-tenth.
The 3- and 5-d-old seedlings were dissected into cotyledons, shoots, and roots prior to extraction. CS, but not BL, was detected in these tissues. The level of 6-deoxoCT and downstream intermediates was high in these tissues. These findings indicate that de novo BR synthesis is already operating in cotyledons, shoots, and roots of the 3- and 5-d-old seedlings. Shoots were found to contain higher levels of CS than roots, in accord with the data reported for Arabidopsis, pea, and tomato (Bancos et al., 2002 ; Symons and Reid, 2004).
The expression of CYP85A1, CYP90B8, CYP92A6, CYP734A11, and LKA was increased as early as during imbibition, whereas the expression of CYP90A9, CYP90A10, CYP85A6, and LKB was elevated in 1-d-old seedlings (Fig. 5; Supplemental Fig. S2). The increase observed in LK was less marked.
Shoots, roots, and cotyledons of 3-d-old seedlings were also examined. It should be noted that expressions of CYP85A1 and CYP85A6 were high in shoots and roots, but scarce in cotyledons (Fig. 6). Expressions of the other genes were rather evenly distributed in shoots, roots, and cotyledons.
The Late C-6 Oxidation Pathway Is Predominant In Pea as Well as in a Majority of Plants
Biosynthesis of CS from campestanol has been known to occur through the early C-6 oxidation pathway via cathasterone or the late C-6 oxidation pathway via 6-deoxoCT, 6-deoxoteasterone (6-deoxoTE), 6-deoxo3DT, 6-deoxoTY, and 6-deoxoCS (Fig. 2; Fujioka and Yokota, 2003
However, we earlier identified BRs belonging to the early C-6 oxidation pathway from pea tissues, although at very low levels, i.e. typhasterol (TY) from immature pea seeds (Yokota et al., 1996
The C-6 oxidation is a rate-limiting step in the production of bioactive BRs because 6-deoxoCS, a direct precursor of CS, commonly accumulates at relatively high levels in several plant species (Nomura et al., 2001
The bas1-D mutant of Arabidopsis, in which CYP734A1 (previously named CYP72B1) encoded by the BAS1 gene is amplified, was reported to deactivate BL and CS by C-26 hydroxylation (Neff et al., 1999
In mature seeds, the biologically active BRs CS and BL were not detected. However, their biologically inactive precursors were stored in mature seeds. Among them, 6-deoxoCT was predominant in mature seeds. This profile of endogenous BRs in mature seeds was not changed after 2 years of storage at 4°C (data not shown), indicating that these precursors may work as storage forms for years. The endogenous BR levels were not affected by imbibition (Table III ), but de novo synthesis of CS and its precursors was clearly observed in 1-d-old seedlings. In contrast, only 6-deoxoCT was drastically reduced in 1-d-old seedlings, indicating that CS and its precursors were synthesized at the expense of 6-deoxoCT stored in seeds. This suggests that 6-deoxoCT is used as a storage BR and that de novo synthesized 6-deoxoCT is not fully supplied at this stage. The level of 6-deoxoCT was largely restored in shoots, roots, and seeds of 3- and 5-d-old seedlings, indicating that de novo synthesis of 6-deoxoCT and upstream intermediates starts within 3 d of the start of germination.
Transcripts of biosynthesis genes LKB, LK, CYP90A9, and CYP90A10, as well as of the receptor gene LKA, were detected in mature seeds, suggesting that these mRNAs may be promptly utilized to generate and perceive BRs as soon as seeds germinate. Interestingly, the level of CYP90A9 mRNA was increased during maturation and reached the highest level in mature seeds (Fig. 4; Supplemental Fig. S1). Furthermore, high levels of LKB and LK were maintained in mature seeds. In accord with this, the LK gene (as well as its Arabidopsis homolog DET2) is known as a steady-state gene whose transcription is little affected by growth stage (Li et al., 1996
In imbibed pea seeds, no change was observed in the profile of endogenous BR levels as compared with that of mature seed. However, the mRNAs of CYP90B8, CYP85A1, and CYP734A11 had already started to increase. In 1-d-old seedlings all mRNAs examined were largely increased (Fig. 5
; Supplemental Fig. S2) and de novo synthesis of CS was observed (Table III). As seen in 3- and 5-d-old seedlings, cotyledons, shoots, and roots were all found to synthesize CS. In 3-d-old seedlings, transcription of CYP85A1 and CYP85A6 was observed in the shoot and root, but was very scarce in the cotyledons (Fig. 6
), reflecting the higher level of CS in shoots and roots than in cotyledons. BL was not detected in any tissues of the dark-grown seedlings in this study, suggesting that only CS is required for early growth of seedlings. However, significant amounts of BL, in addition to CS, have been detected in shoots of light-grown young seedlings (Symons et al., 2002
In tomato, only CS is required for shoot growth, whereas both CS and BL are responsible for the development of fruits (Nomura et al., 2005
Plant Materials We used the pure wild-type cultivar of garden pea (Pisum sativum), Torsdag. Immature seeds were harvested from plants grown in a field under natural conditions in May to June, 2001. Seeds collected were grouped according to their size and appearance (Fig. 1; Table I). The number and total weight (g) of harvested seeds as well as averaged seed weight (g) were as follows (in parentheses): stage 1 (335, 9.7, 0.029), stage 2 (148, 16.3, 0.111), stage 3 (175, 33.0, 0.189), stage 4 (330, 158.2, 0.480; fully expanded, green), stage 5 (10, 4.88, 0.488; fully expanded, yellow), and stage 6 (200, 56.2, 0.281; mature, dry). As for germinating seedlings, 200 mature pea seeds were imbibed in running tap water for 16 h, then incubated in wet paper towels at 25°C in the dark for 1, 3, or 5 d (Fig. 1). The imbibed seeds weighed 409 mg, whereas 1-d-old seedlings weighed 508 mg. Three-day-old seedlings were dissected into cotyledons (459 mg), shoots (110 mg, 13–26 mm), and roots (96 mg, 20– 70 mm). Five-day-old seedlings were also dissected into cotyledons (467 mg), shoots (290 mg, 80–135 mm), and roots (144 mg, 80–125 mm).
The methanol extract of the harvested materials was spiked with 2H6-labeled BRs (Nomura et al., 1999
Primer sequences used in this study are shown in Supplemental Table S2. First primers were designed based on highly conserved nucleotide sequences between legume expressed sequence tags and BR biosynthesis genes of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). PCR amplification was carried out with the Expand High Fidelity PCR system (Roche). Templates were from single-strand cDNA libraries that were made from immature seeds or 7-d-old shoots of pea. The resulting products were sequenced using Long-Read Tower sequencer (Amersham Biosciences). Based on those sequences, gene-specific primers were designed to amplify the 5' and 3' ends of each gene. Primers listed in Supplemental Table S2 were used sequentially for 5'- and 3'-RACE reactions according to the 5'/3' RACE kit (Roche). The resulting products were sequenced and these fragments were assembled to construct an open reading frame. Primers were designed to amplify the full-length cDNA and the full-length clones were sequenced to check the PCR errors. Sequence analysis was performed using MacVector software (Oxford Molecular). The phylogenetic tree was constructed by neighbor-joining with p-distance and bootstrap replication (1,000 replications) using MEGA Version 3.1 software (http://www.megasoftware.net/). Amino acid sequences of the P450 members in Arabidopsis, tomato, and rice were obtained from the Cytochrome P450 home page (http://drnelson.utmem.edu/CytochromeP450.html).
Primer sequences used in this study are shown in Supplemental Table S3. The transcript levels of genes were analyzed by semiquantitative reverse transcription (RT)-PCR. Total RNA was extracted from pools of 10 to 20 immature seeds harvested from five to 10 pods and from shoots, roots, and cotyledons excised from pools of 10 to 40 individuals of 3- and 5-d-old seedlings using RNeasy Plant Mini kit with RNase-Free DNase set (Qiagen). Single-strand cDNA was synthesized using 2.5 µg of total RNA, SuperScript II reverse transcriptase (Invitrogen), and oligo(dT) primer according to the manufacturer's instructions. PCR amplification was performed using 500 nM gene-specific primers and 50 to 100 nM actin primers (as a control) with the same concentration of template cDNAs in the same tube. The product was sequenced to confirm the identical band. RT-PCR products obtained by three different amplification cycles were run on an agarose gel. Gels stained with ethidium bromide were digitized and analyzed using luminescent image analyzer GENEGENIUS (Syngene). Sequence data from this article have been deposited with the DDBJ/EMBL/GenGank data libraries under the following accession numbers: AB218761 (CYP90A9), AB218762 (CYP90A10), AB218763 (CYP90B8), AB277551 (CYP90D7), and AB218764 (CYP734A11).
The following materials are available in the online version of this article.
We thank Kyomi Shibata for technical assistance. We also thank Prof. James B. Ried (University of Tasmania, Australia) for originally providing pea seeds and critical reading of the manuscript, and Dr. David Nelson (University of Tennessee) for the P450 designation. Received November 15, 2006; accepted February 16, 2007; published February 23, 2007.
1 This work was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science (grant no. 1146007 to T.Y.) and by a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists (to T.N.). 
2 Present address: Plant Science Center, RIKEN, Yokohama 230–0045, Japan. 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: Takao Yokota (yokota{at}nasu.bio.teikyo-u.ac.jp).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093096 * Corresponding author; e-mail yokota{at}nasu.bio.teikyo-u.ac.jp; fax 81–28–627–7187.
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-Brassinosteroid dehydrogenase activity in Arabidopsis and tomato. Phytochemistry 58: 989–994
