Genes & Genomics (2010) 32: 309-317 DOI 10.1007/s13258-010-0011-z
RESEARCH ARTICLE
Identification and characterization of SHI family genes from Brassica rapa L. ssp. pekinensis Joon Ki Hong · Jung Sun Kim · Jin A Kim · Soo In Lee · Myung-Ho Lim · Beom-Seok Park · Yeon-Hee Lee 1)
Received: 3 February 2010 / Accepted: 22 April 2010 / Published online: 31 August 2010 © The Genetics Society of Korea and Springer 2010
Abstract SHI (short internodes) is a negative regulator of gibberellin-induced cell elongation. Extensive searches in the Brassica rapa genome allowed for the prediction of at least six different SHI-related genes on six chromosomes in the genome. Genome structural examination revealed that these genes had one intron each in their corresponding open reading frames. Protein structure comparisons using the CLUSTALW program and based on alignments of all BrSRS (B. rapa SHI-related sequence) proteins revealed broad conservation of the RING finger-like zinc finger and IGGH motifs. According to the phylogenetic relationship based on deduced amino acid sequences, the six BrSRS proteins were most closely related to Arabidopsis SRS (AtSRS) proteins; however, BrSRS proteins were dispersed in the phylogenetic tree. Semi-quantitative RT-PCR analysis indicated that the six BrSRS genes exhibited different expression patterns in various tissues and responded differently to growth phytohormones. The differences among the six BrSRS genes with respect to gene structure and expression pattern suggest that these genes may play diverse physiological roles in the developmental process of B. rapa.
Keywords Gene structure; Negative regulator; Phylogenetic relationship; Semi-quantitative RT-PCR; SHI-related sequence; Short internodes
J.K. Hong · J.S. Kim · J.A. Kim · S.I. Lee · M.‐H. Lim · B.‐S. Park · Y.‐H. Lee ( ) National Academy of Agricultural Science, Rural Development Administration, Suwon 441‐707, Korea e-mail:
[email protected]
Introduction SHI (short internodes) acts as a negative regulator of responses of the plant hormone gibberellin (GA) through transcriptional control when activated in plants (Fridborg et al., 2001). Several investigators have annotated and characterized some members of the SHI/STYLISH (STY) gene family in the model angiosperm Arabidopsis thaliana/Oryza sativa and the bryophyte model Physcomitrella patens (Fridborg et al., 2001; Kuusk et al., 2002, 2006). This gene family is plant-specific because no homologs have been found outside the plant kingdom (Fridborg et al., 2001). The SHI-related proteins have very similar organization and share two conserved domains:a putative RING finger–like zinc finger as a C3HC3H RING domain and an IGGH domain of unknown function (Fridborg et al., 2001). The RING domain of SHI-related proteins is similar to the consensus of the zinc-binding C3HC4 RING finger motif (Freemont, 1993; Lovering et al., 1993), which generally mediates protein–protein interactions in numerous otherwise unrelated proteins in different eukaryotes (Borden 2000). A short acidic cluster is present in the IGGH domain in several of SHI-related proteins. In addition to these consensus sequences, the SHI-related proteins also share a nuclear localization signal and various glutamine-rich (Q-rich) regions that are commonly found in transcriptional regulatory proteins (Mitchell and Tjian, 1989). SHI, a putative transcription regulator, modifies the transcription of genes encoding products that either promote or inhibit the GA response (Fridborg et al.,2001; Olszewski et al., 2002). SHI-related genes may regulate premature growth or development by controlling GA responses. In Arabidopsis, the SHI gene expressed in young organs prevents inappropriate elongation growth in responses to GA (Fridborg et al., 2001; Olszewski et al., 2002). Additionally, overexpression of the SHI gene in barley aleurone cells reduces α-amylase ex-
310
pression by GA, indicating that SHI can negatively regulate the GA response in heterologous species (Fridborg et al., 2001; Olszewski et al., 2002). Recently, phenotypic analysis of plants with mutations in one or several SHI family genes revealed that these mutations affect the development of the gynoecium, the female reproductive structure of the flower, as well as leaf development and organ identity in floral whorls (Kuusk et al., 2002, 2006). In addition, multiple mutant analyses have shown that many SHI-related genes are partially redundant in function and regulate the fundamental features of plant development, such as development of the gynoecium, stamen, and leaf, in a dose-dependent manner (Kuusk et al., 2006; Sohlberg et al., 2006). In particular, STY1 and related genes are expressed in the apical region of the developing gynoecium, and changes in gene expression affect apical–basal patterning of gynoecium morphogenesis through the regulation of auxin homeostasis (Smyth et al., 1990; Sohlberg et al., 2006). This indicates that STY1 and related SHI genes affect various developmental processes by regulating auxin homeostasis (Sohlberg et al., 2006). Thus, SHI family members may act in concert with the growth phytohormones GA and auxin in plant developmental process. In spite of the importance of Arabidopsis SHI and SHI-related genes in the control of development and signal transduction in response to growth phytohormones, the function of individual genes in other species has not yet been characterized. In this study, we identify and characterize six new SHI-related genes in B. rapa. We first annotate SHI family genes in B. rapa and predict the exon–intron organization of these genes using bioinformatics resources. In addition, we provide evidence from semi-quantitative reverse transcription (RT)PCR analysis that BrSRS (B. rapa SHI-related sequence) genes have different expression patterns in various tissues and in response to growth phytohormones.
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index.jsp; Yang et al., 2005). Exon–intron splice sites were analyzed and confirmed by comparing the B. rapa BAC clones and cDNA sequences with GENSCAN (http://genes.mit.edu /GENSCAN.html; Burge and Karlin, 1997) and GeneMark (http://opal.biology.gatech.edu/GeneMark/; Lukashin and Borodovsky, 1998). The BrSRS proteins and the other SRS deduced amino acid sequences derived from cDNA or genomic DNA were obtained from TAIR, RGP (http://rgp.dna.affrc.go. jp/), Populus genome assembly 1.0 (http://genome.jgi-psf.org/ Poptr1/Poptr1.home.html), NCBI (http://www.ncbi.nlm.nih. gov), and the BrGP database. Analysis of DNA and comparison of deduced amino acid sequences were performed with current bioinformatics tools. Sequence alignment was performed using the GeneDoc program (http://www.pcs.edu/biomed/genedoc). The phylogenetic tree was created with Molecular Evolutionary Genetic Analysis (MEGA) software, version 4.0 (http://www. megasoftware.net) using the neighbor-joining method. Complete deletion and Poisson correction were used for treatment of amino acid gaps and substitution models on the SRS multiple alignments (Kumar et al., 2004). The sampling variance of the distance values was estimated from a 1,000 bootstrap resampling of the alignment columns. The molecular masses of the six BrSRS proteins were calculated using the BioEdit sequence Alignment Editor Program (Isis Pharmaceuticals, USA). Genetic positioning of BrSRS genes Sequence-based genetic mapping was conducted to localize six BAC clones containing the BrSRS genes on the reference map of the B. rapa genome using simple sequence repeat, intron based-polymorphism, and EST markers. The BAC clones harboring BrSRS genes were genetically localized on the JWF3p mapping population (Kim et al., 2006).
Materials and Methods
Plant materials and semi-quantitative RT-PCR analysis
Sequence data, alignment, and phylogenetic analysis
B. rapa L. ssp. pekinensis cv. JangWon seeds were grown on MS medium (Murashige and Skoog, 1962) containing 3% sucrose and 0.25% phyta-gel (pH 5.8) and stratified at 4℃ for 5 days in darkness to induce synchronous germination. The plants were grown at 23℃ for 2 weeks under long-day conditions (16 h light and 8 h dark) as described by Yang et al. (2006), then transplanted to soil. The plants were transferred to a growth chamber, and samples were collected throughout the sampling period. Total RNA was extracted from B. rapa seedlings and tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA). For semi-quantitative RT-PCR analysis, cDNA was synthesized from 3 ㎍ total RNA using MMLV reverse transcriptase
Searches for B. rapa SRS genes were performed in publicly available genome databases. To assemble the SRS genes in B. rapa, we adopted an in silico approach following Zang et al. (2009) with minor modifications. Sequences of Arabidopsis SHI family genes obtained from The Arabidopsis Information Resource database (http://www.arabidopsis.org) were used to search B. rapa homologs from the sequence database of bacterial artificial chromosome (BAC) clones of B. rapa L. ssp. pekinensis cv. Chiifu using tblastx at NCBI (http://blast. ncbi.nlm.nih.gov/Blast.cgi) and the Brassica rapa Genome Project (BrGP) database (http://www.brassica-rapa.org/BRGP/
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Growth phytohormone treatments
Table 1. Oligonucleotide primers used in RT-PCR. Target genes BrSTY1
Expected size (bp)
Primer sequences 5'-TGAATGGCGGGTTTTTTCTCGTTAG-3'
To evaluate the inducibility of expression of BrSRS genes in response to growth phytohormones, we subjected 2-week-old plants to treatment with abscisic acid (ABA), gibberellic acid (GA), naphthaleneacetic acid (NAA), or 6-benzylaminopurine (BA). These plants were then grown hydroponically in MS solutions containing 10 μM ABA (mixed isomers; Sigma, St. Louis, MO, USA), 10 μM BA (Fluka, Buchs, Switzerland), 10 μM GA3(Duchefa, Haarlem, The Netherlands), 10 μM NAA (Sigma), or without phytohormones. After incubation for 6 hr at 23℃ the whole plants and shoot apical meristems were harvested in liquid nitrogen and stored at -80℃ until total RNA was extracted. The expression pattern of BrSRS genes in response to growth phytohormones was analyzed by semi-quantitative RT-PCR, as described previously.
1184
5'-TGTCAGATTTCCATCATGATCTTGG-3' BrLRP1
5'-AACCTACCGATGTCGGATTCCGGTG-3'
1281
5'-TATGGTTTAACTGTAAAACCCAGC-3' BrSTY2a
5'-AGTGTTAGGTATTAGAAAATGTCTG-3'
1177
5'-TCTTGATTATTTAAGATCTTGGTGG-3' BrSTY2b
5'-AGTGTTAGGTCTTAGGAAATGGCTG-3'
1128
5'-TCTTGATTTAATCAAGATCTTGGTG-3' BrSRS5
5'-TGGAAAATAAATGGCAGGATTTTTC-3'
1240
5'-TTCAATAACATTGTCCCTCGGAGG-3' BrSRS7
5'-AGTAGAAGAAATGGCTGGATTGTTC-3'
1213
5'-ATCTTAAGACCTAGGAGATGCAAAG-3' Bactin
5'-TGGCATCACACTTTTCTACAA-3'
515
5'-CAACGGAATCTCTCAGCTCC-3'
Results and Discussion
(RNaseH free; Toyobo, Osaka, Japan), according to the manufacturer’s instructions. Each cDNA sample was diluted 1 : 3, and 2 μℓ diluted cDNA was used for PCR amplification (94℃ for 1 min, 57℃ for 1 min, 72℃ for 1 min) in a volume of 50 μℓ with a gene-specific primer set (Table 1). Bactin was used as the internal control for RNA quantity (Yao et al., 2005; Hong et al., 2008). The resulting PCR products (5 μℓ each) were analyzed using electrophoresis and ethidium bromide staining. All PCR products were sequenced and matched to individual gene sequences (data not shown). The relative expression levels were analyzed by RT-PCR amplification using the Quantity One program (Bio-Rad, Hercules, CA, USA; Liang et al., 2009).
Identification and genomic organization of B. rapa SHI family genes To assemble a complete and non-redundant sequence set of BrSRS gene sequences from B. rapa, we used an in silico approach by first screening the genome sequences of B. rapa (Hong et al., 2008; Zang et al., 2009). Full-length sequences of 10 Arabidopsis SHI family genes (SHI, STY1, STY2, LRP1, and SRS3-SRS8) containing the conserved consensus sequences and motifs (the RING finger–like zinc finger motif and the IGGH domain) were blasted to the sequenced BAC clones derived from B. rapa L. ssp. pekinensis cv. Chiifu using tblastx in the BrGP and NCBI B. rapa databases. Blast searches were repeated with each new BrSRS gene sequence found
Table 2. AtSHI gene family members and number of BAC clones selected from the B. rapa genome database.
a
a
Arabidopsis gene name
AGI number
Selected BAC clone
BAC Acc. No.
B. rapa gene name
CDS Acc. No.
AtSHI
At5g66350
-
-
-
-
AtSTY1
At3g51060
KBrB088I08
AC189508
BrSTY1
GU205263
AtSTY2
At4g36260
KBrB048F07 KBrH109I16
AC189373 Unpublished
BrSTY2a BrSTY2b
GU205264 GU205265
AtLRP1
At5g12330
KBrB041J18
AC189340
BrLRP1
GU205266
AtSRS3
At2g21400
-
-
-
-
AtSRS4
At2g18120
-
-
-
-
AtSRS5
At1g75520
KBrH012I05
AC189586
BrSRS5
GU205267
AtSRS6
At3g54430
-
-
-
-
AtSRS7
At1g19740
KBrH006P24
AC189558
BrSRS7
GU205268
AtSRS8
At5g33210
-
-
-
-
BAC clones were selected from the BrGP database.
312
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Figure 1. Genomic organization of the BrSRS genes. Exons are indicated by boxes and introns by lines. The putative zinc-binding RING finger, nuclear localization signal, and IGGH domains are represented as gray, cross-hatched, and black boxes, respectively. The number of base pairs corresponding to each intron is indicated in the predicted structure. The number of amino acids encoded by the open reading frames, the molecular mass (kDa), and the presence of a predicted nuclear localization signal (NLS) are also indicated.
in the databases to complete BrSRS gene collection. All selected BAC clones were annotated using GENSCAN and GeneMark. Six non-redundant BrSRS sequences were found in the sequences of six BAC clones (Table 2), and the predicted BrSRS genes were dissected from the BAC clones for further analysis. The genome structure and chromosome locations of the BrSRS genes were determined from the BAC clone sequences of B. rapa (Fig. 1 and Fig. 2). Comparing the cDNA sequences and the BAC clone sequences established that all genes consisted of two exons and encoded putative proteins of 314-380 amino acids with calculated molecular masses of 33.7-39.3 kDa proteins contained a putative nuclear localization signal (Fig. 1). Although their lengths varied, introns were present between sequences encoding the conserved RING finger-like zinc finger motif and IGGH domains. These results reveal that the genomic organization of BrSRS genes shows significant similarity in exon-intron structure to that of SRS genes reported from Arabidopsis (http://www.arabidopsis.org; Fridborg et al., 1999). Because the BAC clones containing BrSRS genes are linked to genetic markers, it was possible to estimate the relative position of the sequences in the genetic map (Kim et al., 2006). The six BrSRS genes were distributed on 6 out of the 10 B. rapa chromosomes (Fig. 2). By comparing their positions in relation to large segmental duplication events identified from the BrGP database and Kim et al. (2006), we found that BrSTY2a and BrSTY2b constituted a duplicate paralogous gene pair (data not shown). In fact, the Brassica genomes contain evolutionarily triplicated counterparts of corresponding region of the A. thaliana genome. However, the number of genes in the Brassica genome is increased approximately two-fold compare with that of the A. thaliana genome because the B. rapa genome shrinkage after genome triplication (Lysak et al., 2005; Yang et al., 2005; Park et al., 2008; Mun et al., 2009). Therefore, the actual
Figure 2. Chromosomal distribution of BrSRS genes in the B. rapa genome. All positions in centMorgans (cM) were estimated in accordance with genetic markers assigned to the BAC clones. Centromeres are represented by black boxes.
number of BrSRS genes could be estimated to be 20 although we identified six BrSRS genes due to the limited information of the sequenced BAC clones in this study (Park et al., 2008; Mun et al., 2009). Comparison and phylogenetic analysis of BrSRS proteins The alignment of the deduced amino acid sequences of BrSRS proteins with AtSHI and AtLRP1 proteins from Arabidopsis is shown in Figure 3. The pairwise identity among the mature deduced amino acid sequences of the six BrSRS proteins varied from 29.3 to 88.7%, indicating that the BrSRS gene family is quite divergent (Table 3). Despite being highly divergent in amino acid sequences, the predicted proteins showed particularly high sequence identity over two regions (Table 4). The first was positioned in the N-terminal region of proteins, and sequence identity varied between 80.4 and 97.0% (Table 4, RING domain). A cysteine/histidine-rich stretch with consensus sequence Cys-X2-Cys-X7-Cys-X-His-X2-Cys-X2-CysX7-Cys-X2-His (X denotes any amino acid) or C3HC3H was present in almost all BrSRS family members (Fig. 3A). These consensus sequences were similar to the previously described
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313
Table 3. Pairwise sequence identity values for six BrSRS proteins based on their deduced amino acid sequences.
Table 4. Pairwise amino acid sequence identities in the RING and IGGH domains of BrSRS proteins.
BrSTY1 BrLRP1 BrSTY2a BrSTY2b BrSRS5 BrSRS7 BrSTY1 BrLRP1 BrSTY2a BrSTY2b BrSRS5 BrSRS7
-
Pairwise % identity of RING domain
30.3
49.0
51.4
38.3
36.9
-
33.7
32.6
29.3
29.8
BrSTY1
80.4
82.6
82.6
87.0
89.1
-
88.7
36.5
37.6
BrLRP1 58.7
-
80.4
80.4
80.4
84.8
-
36.5
39.1
BrSTY2a 65.2
58.7
-
97.0
80.4
84.8
68.3
BrSTY2b 67.4
60.9
95.8
-
80.4
84.8
-
BrSRS5
82.6
63.0
72.9
72.9
-
95.7
BrSRS7
76.1
54.3
68.1
68.1
89.4
-
-
zinc-binding C3HC4 RING finger motif (Freemont, 1993; Borden, 2000; Fridborg et al., 2001). As can be observed in Figure 3A, BrLRP1 differed from other family members in that it lacked the first conserved His residue of the C3HC3H RING sequence, and the sequence showed similarity to the consensus sequence of AtLRP1. Furthermore, a putative nuclear localization signal was found immediately downstream of the putative RING domains of the BrSRS proteins (Fig. 3A), which are basic stretches that conform to the consensus of the typical nuclear localization signal defined as four arginines (R) and lysines (K) within a region of six amino acids (LaCasse, 1995; Boulikas, 1994; Fridborg et al., 1999). The second consensus sequence element, an IGGH domain of unknown function, was located in the C-terminal region of all BrSRS family proteins (Fig. 3B), and the sequence identity varied between 58.7 and 95.8% (Table 4, IGGH domain). This domain showed no distinguishable sequence similarity to any previously described protein motifs (Fridborg et al., 2001; Kuusk et al., 2002). A short stretch of acidic residues was present upstream of the IGGH domain in the BrSRS proteins. These sequence features indicate that BrSRS proteins function as transcriptional regulators in plants (Mitchell and Tjian, 1989). Putative SHI homologs have been found in other plant species, as revealed by database searches of Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Physcomitrella patens, but are absent in any organization outside the plant kingdom. These sequences can be used to identify SHI-related proteins from gene or protein databanks and to distinguish them from other member proteins containing zinc-binding RING finger motifs. Taken together, these data suggest that BrSRS proteins may function in transcriptional regulation (Mitchell and Tjian, 1989). To evaluate the phylogenetic relationships between the BrSRS proteins and other SRS proteins, we compared the deduced amino acid sequences of SHI-related proteins identified in Arabidopsis, rice, Populus, and Physcomitrella and generated a phylogenetic tree with the MEGA analysis platform using the neighbor-joining method (Fig. 4). The bootstrap values for the main subgroup were higher than 50%, suggesting
BrSTY1 BrLRP1 BrSTY2a BrSTY2b BrSRS5 BrSRS7 -
Pairwise % identity of IGGH domain
that they presented true clusters of SHI-related proteins sharing a similar origin. The phylogenetic analysis indicated that the BrSRS proteins, although probably not representing the total number of SHI-related proteins of the B. rapa genome, were most closely related to Arabidopsis SHI-related proteins, indicating that Arabidopsis and B. rapa proteins are probably functional homologues. However, BrSRS proteins and AtSHI-related proteins were dispersed in the tree, which made it difficult to determine the orthologous partners in rice and Physcomitrella. The alignment and phylogenetic analysis strongly suggest that divergent BrSRS in B. rapa might differ in their distinct cellular functions. In fact, Kuusk et al. (2006) suggested that, despite being highly divergent in sequence, many SHI-related proteins are partially redundant in function but show evidence of sub-functionalization.
Figure 3. Comparison of amino acid sequences of BrSRS proteins. Alignment of the deduced amino acid sequences of six BrSRS proteins with AtSHI and AtLRP1 proteins is shown. A, Sequence comparison of the zinc finger RING domains of the SRS proteins. Asterisks indicate conserved Cys and His zinc ligand residues in the RING finger motifs. The box indicates putative nuclear localization signals. Identical and similar residues are displayed in black and gray boxes, respectively. B, Comparison of the IGGH domain of SRS proteins. Acidic stretches (black box) and four IGGH residues (white box) are indicated. The GenBank accession numbers for these sequences are as follows: Arabidopsis thaliana, AtSHI (A5g66350), AtLRP1 (At5g12330); Brassica rapa, BrSTY1 (ADA60972), BrSTY2a (ADA60973), BrSTY2b (ADA60974), BrLRP1 (ADA60975), BrSRS5 (ADA60976), BrSRS7 (ADA60977).
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Expression of BrSRS genes in various tissues of B. rapa
Figure 4. Phylogenetic relationships between BrSRS proteins and other SRS proteins. A phylogenetic tree was generated based on neighbor-joining analysis of the predicted amino acid sequences of 6 BrSRS proteins and 24 other SRS proteins (9 from Arabidopsis, 5 from rice, 9 from Populus, and 1 from Physcomitrella). Complete deletion and Poisson correction methods were used for the treatment of amino acid gaps and substitution models on the SRS multiple alignments. Bootstrap values are indicated as percentages. The length of each pair of branches represents the phylogenetic distance between sequence pairs. BrSRS proteins are marked with an asterisk. The scale bar corresponds to 0.05 amino acid substitutions per residue. The gene accession numbers for these sequences are as follows: Arabidopsis thaliana, AtSHI (At5g66350.1), AtLRP1 (At5g12330.1), AtSTY1 (At3g51060.1), AtSTY2 (At4g36260.1), AtSRS3 (At2g21400.1), AtSRS4 (At2g18120.1), AtSRS5 (At1g75520.1), AtSRS6 (At3g54430.1), AtSRS7 (At1g19790.1); Brassica rapa, BrSTY1 (ADA60972), BrSTY2a (ADA60973), BrSTY2b (ADA60974), BrLRP1 (ADA60975), BrSRS5 (ADA60976), BrSRS7 (ADA60977); Oryza sativa, OsSRS1 (OJ1254 _E07.16), OsSRS2 (OJ1112_E06.1), OsSRS3 (B1139B11.6), OsSRS4 (P0712G01.5), OsSRS5 (OJ1286_E05.15); Populus trichocarpa, PtSRS1 (fgenesh1_pg.C_LG_IX000438), PtSRS2 (eugene3.00041233), PtSRS3 (eugene 3.00570131), PtSRS4 (eugene 3.00020259), PtSRS5 (fgenesh1_ pg.C _LG_V001495), PtSRS6 (fgenesh1_pg.C_LG_I001988), PtSRS7 (eugene 3.00090945), PtSRS8 (fgenesh1_pg.C_LG_III001700), PtSRS9 (fgenesh1_pg.C_LG_I000246); Physcomitrella patens, PpSRS1 (AAX53173).
Previously, the spatial and temporal expression patterns of SHI, STY1, STY2, SRS5, and LRP1 in Arabidopsis had been investigated using reporter constructs, in situ hybridization, and RT-PCR (Smith and Fedoroff, 1995 Fridborg et al., 2001; Kuusk et al., 2002, 2006). AtLRP1 transcripts were detected in flowers and roots (Smith and Fedoroff, 1995). AtSHI, AtSTY1, AtSTY2, and AtSRS5 showed overlapped expression in lateral root primordia, young rosette leaves, hydathodes, and the flower, but these genes showed some temporal as well as spatial divergences (Fridborg et al., 2001; Kuusk et al., 2002, 2006). However, the expression patterns of AtSRS3, AtSRS4, AtSRS6 and AtSRS7 were largely unknown. Thus, in order to precisely define the spatial and temporal expression pattern of BrSRS genes in B. rapa, we examined tissue-specific expression of six BrSRS genes with semi-quantitative RT-PCR using total RNA extracted from seeds, seedlings, roots, leaves, shoot apical meristems, flower buds, flowers, stems, and siliques. The Bactin gene of B. rapa was used as an internal control to adjust the amount of cDNA for PCR because Bactin is constitutively expressed in all tissue types (Yao et al., 2005 Hong et al., 2008). As shown in Figure 5, all of the BrSRS genes were expressed in shoot apical meristems at variable relative intensity, underlying the predominant role of SHI-related genes in these regions. However, the genes showed spatial and temporal differences in other tissues. BrSTY1 had the highest expression in shoot apical meristems and was also detected in seedlings, flower buds, flowers, leaves, and roots. BrLRP1 revealed a rather specific expression pattern: It was strongly expressed in leaves and weakly detected in roots and stems. BrLRP1 also increased slightly in seedlings, whereas BrSTY1, BrSTY2a, BrSTY2b, and BrSRS7 decreased during seedling growth. BrSTY2a and BrSTY2b had similar amino acid sequences and were tightly clustered in the phylogenetic tree but were expressed differently. BrSTY2a was abundantly expressed in seedlings, whereas BrSTY2b accumulated to high levels in flower buds, although it was also detected in seedlings and roots. BrSRS5 was detected in seedlings. BrSRS7 was expressed ubiquitously at different relative intensities in all organs except 0-day-old seedlings. Thus, the specific expression pattern of BrSRS genes in various tissues indicates their differential impact on developmental process and could be the result of a process of sub-functionalization (Papp et al.,2003; Veitia, 2004, 2005; Kuusk et al., 2006). However, overlapping expression in shoot apical meristems suggests that BrSRS genes might also act redundantly to regulate developmental processes in shoot apical meristems (Kuusk et al., 2006). Overall, these data indicate that the expression of six BrSRS genes is tissue-specific
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Figure 5. Semi‐quantitative RT‐PCR analysis of BrSRS genes during seedling growth and in different tissues. Total RNA was isolated from seedlings (lanes 0d‐7d), roots (lane R), leaves (lane L), shoot apical meristems (lane Sa), flower buds (lane Fb), flowers (lane F), inflorescence stems (St), and siliques (Si). A 3‐㎍ aliquot of total RNA was reverse‐transcribed into first‐strand cDNA for semi‐quantitative RT‐PCR analysis of gene expression (upper panel). Bactin was used as an internal control for RNA quantity. Relative expression levels calculated using the Quantity One program (lower panel). Data means ± S.E. from at least three independent experiments.
and developmentally regulated. Regulation of BrSRS family genes by growth phytohormones in B. rapa To analyze the responsiveness of BrSRS genes to growth phytohormones, we examined gene expression using semi-quantitative RT-PCR in 2-week-old whole plants of B. rapa with various growth phytohormones: 10 μM ABA, BA, GA3, and NAA. As shown in Figure 6A, the expression of BrSTY1, BrSTY2a, BrSRS5, and BrSRS7 transcripts in whole plants increased at variable relative intensities in response to these four growth phytohormones, whereas expression of BrLRP1 increased slightly in response to BA and GA3. However, the BrSTY2b transcript was expressed at nearly consistent levels following all growth phytohormone treatments examined (Fig. 6A). These data suggest that the BrSRS genes may be involved in developmental processes mediated by growth phyto-
315
Figure 6. Semi‐quantitative RT‐PCR analysis of BrSRS genes in response to growth phytohormone treatment. Total RNA was isolated from 2‐week‐old whole plants (A) and shoot apical meristems (B) of B. rapa exposed to 10 μM ABA, 10 μM BA, 10 μM GA3, 10 μM NAA, or without phytohormones, as indicated (see Materials and Methods). A 3‐㎍ aliquot of total RNA was reverse‐transcribed into first‐strand cDNA for semi‐quantitative RT‐PCR analysis of gene expression (upper panel). Bactin was amplified as a control. Relative expression levels measured using Quantity One (lower panel). Data means ± S.E. from at least three independent experiments.
hormones in plant cells. During development, phytohormones and transcription factors cooperate to balance meristem maintenance and organ production (Fleet and Sun, 2005; Shani et al., 2006). We considered the possibility that expression of BrSRS genes might be affected by growth phytohormones in shoot apical meristem maintenance and organ initiation. Therefore, we used semi-quantitative RT-PCR to analyze the expression pattern of BrSRS genes in shoot apical meristems treated with 10 μM ABA, BA, GA3, or NAA. Figure 6B shows that expression of all BrSRS genes in shoot apical meristems in response to GA3 was reduced, indicating that all BrSRS genes are negatively affected by GA in shoot apical meristems. A 6-h treatment of shoot apical meristems with phytohormones resulted in highly elevated expression of BrSTY1 and BrSRS5 in the presence of ABA, cytokinin (BA), and auxin (NAA). The BrLRP1 transcript was stimulated by BA but was not detected after NAA treatment. BrSTY2a was induced in response to NAA but was decreased by ABA. BrSTY2b gene expression was strongly activated in response to BA and NAA. However, the level of BrSRS7 transcript decreased following all of the
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growth phytohormone treatments examined (Fig. 6B). These data indicate that expression of BrSRS genes is differently affected by growth phytohormones in shoot apical meristems. This means that BrSRS genes can modulate phytohormonal regulation of developmental processes in a tissue-specific manner in B. rapa. Also, the overlapped gene expression in response to growth phytohormones suggests that BrSRS genes may act redundantly in the same phytohormone response pathway in a dose-dependent manner (Kuusk et al., 2003). In this study, we described the identification and characterization of SHI-related genes in B. rapa. The specific expression patterns of the genes according to different tissues and phytohormones treatments suggest that these genes may play diverse physiological roles in response to complex developmental cues in B. rapa. Further investigation of BrSRS genes will help to determine the biological roles and function of SHI-related genes in plant development, growth, and response to growth phytohormones. Acknowledgements This work was supported by grants from the National Academy of Agricultural Science (200901FHT020711365), the Rural Development Administration, Republic of Korea. J.K. Hong was supported by a 2009 Post Doctoral Course Program from National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.
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