Introduction
Root growth regulation is highly important for plants and is controlled by plant hormones. Brassinosteriod (BR) is one of the important hormones of plants. It participates in the regulation of a large variety of developmental processes, from seed germination to cell elongation, fruit ripening, organ senescence, abscission, and stress protection (
Mandava, 1988). More and more scientists have recognized the importance of BRs during plant development and identified genes involved in BR biosynthesis and signal transduction. Many groups are beginning to study other related genes involved in BR signal transduction using genetic screening in
Arabidopsis. Up to now, the basic pathway of BR signal is clear already in
Arabidopsis. In contrast with the rapid progress in understanding how the plant steroid signals reach the nucleus to regulate gene activities, little is known about how BRs induce nongenomic responses at the cell membrane or in the cytosol.
Proteomics is used to analyze gene products in various tissues and physiologic states of cells (
Pandey and Mann, 2000), and it has become very important in the functional genomic field now that several genomes have been sequenced completely and analytical methods for protein characterization have been developed. Progress in plant proteomics has largely been made possible by two-dimensional (2D) gel electrophoresis-based proteomic approaches (
van Wijk, 2001).
In this study, after BR treatment, the total protein from rice root, rice suspension cells, and two BR-related rice mutants was isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS). In an attempt to identify the proteins involved in BR response, we evaluated BR-induced changes in the isolated proteins. 2D gel analysis revealed that 33 proteins were either upregulated or downregulated by BR treatment. Of them, the actin depolymerizing factor, glycine-rich RNA binding proteins, and IAA amidohydrolase may play important roles in the BR-regulated effect. As a result, we concluded that the proteomic approach may be an effective means of identifying novel proteins implicated in hormone response.
Materials and methods
Plant growth assay
When the root length grew to 1 cm, germinated rice seeds (Oryza sativa L. japonica) were sown in basins filled with liquid culture medium of 0.1, 1.0, 10, 100, and 1000 nmol/L BL separately. The culture medium containing the same volume of 80% ethanol was used as the control. These basins were put in the greenhouse with the medium exchanged everyday to keep the culture medium invariable. Seven days later, the seedlings were removed from each basin, and the root and shoot lengths were measured.
Plant material and growth condition
Rice seedlings were cultured with liquid culture medium and maintained in the culture box. Seven-day-old seedlings were transplanted into the liquid culture medium with 0.1 nmol/L and 1000 nmol/L BL separately. The culture medium of 80% ethanol was used as the control. Root was taken after being treated for 6 h to extract proteins and RNA.
Protein extraction
About 0.1 g root powder (ground under liquid nitrogen) was weighted in a tube, added with 1 mL 10% trichloroacetic acid/acetone, stored at -20°C overnight, and centrifuged at 14000 r/min for 20 min at 4°C. After removing the supernatant and washing the pellet twice in 1 mL ice-cold acetone, the acetone was removed away as possible. Thereafter, the pellet was suspended in 100 µL lysis buffer containing 9 mol/L urea, 4% (w/v) CHAPS, 2% ampholine at pH 3-10 (Amersham Biosciences), and 1% DTT, incubated for 2 h at room temperature and pipetted up and down to dissolve the proteins. Then, it was incubated at 30°C for 1 h and spun in Eppendorf microcentrifuge at 4°C for 20 min at 14000 r/min to obtain the supernatant, which was used to determine protein concentration and identify proteins by 2D-PAGE.
RNA extraction
RNA extraction was conducted by grinding 0.1 g tissue in liquid nitrogen, transferring the powder into 1.5 mL tubes containing 1 mL Trizole, and adding 200 µL chloroform to each tube, which was centrifuged at 12000 r/min for 5 min at 4°C. When aqueous phases were carefully pipetted into clean screw-cap centrifuge tubes (interphase and lower phase used to extract protein), equal volume isopropanol was added to each of the aqueous phase, covered, and mixed by gentle inversion. After centrifugation at 12000 r/min at 4°C for 5 min, the supernatant was discarded, and the pellet was washed twice with 700 µL of 75% ethanol by votexing briefly and then recentrifuged at 12000 r/min at 4°C for 2 min. Moreover, after discarding the supernatant and briefly drying the pellet (5-10 min; not longer), 20 µL DEPC-H2O was added.
Gel electrophoresis
Prepared samples were separated in the first dimension by isoelectric focusing (IEF) and in the second dimension by sodium dodecyl sulfate (SDS)-PAGE. The protein with its concentration determined was diluted with rehydration buffer of 9 mol/L urea, 4% CHAPS, 0.5% Ampholines (pH 3.0-10.0), 1% DTT, and 0.002% Bromophenol Blue. IPG electrophoresis was carried out at 0 V for 12 h, followed by 500 V for 1 h and 1000 V for 1 h, and 8000 V for 9.5 h. After IPG, SDS-PAGE in the second dimension was performed using 15% polyacrylamide gel. The gels were stained with Coomassie Brilliant Blue (CBB), and the image analysis was performed. Images of 2D-PAGE were synthesized, and the positions of individual proteins on the gels were evaluated automatically using ImageMaster 2D Elite software (Amersham Biosciences). Protein spots whose intensity in the BR-treated medium was altered relative to the untreated control medium were quantitatively analyzed. In addition, those spots whose abundance was varied reproducibly greater than twofold were considered for identification by MALDI-TOF MS.
MALDI-TOF MS and database searching
For protein identification by MALDI-TOF MS, one aliquot of the enzyme digest solution was spotted onto a sample plate with matrix (α-cyano-4-hydroxcinnamic acid, 8 mg/mL in 50% v/v TFA) and allowed to air dry. MALDI-TOF MS acquisition was an Autoflex MALDI mass spectrometer (Bruker Daltonics, Germany), equipped with a flight tube (reflex mode, 2.6 m long), laser (337 nm), and scout 384 targets system. Accelerating voltage was 20 kV and microchannel plate (MCP) detector worked at 1.6 kV. Mass spectra were acquired in positive mode. Known trypsin autocleavable peptide masses (906.51 Da and 2273.16 Da) were used for a two-point internal calibration for each spectrum. Peptide mass fingerprinting (PMF) was searched against NCBI protein databases using the search engine Matrix Science at http://www.matrixscience.com. Oryza sativa was chosen for the taxonomic category. All peptide masses were assumed as monoisotopic [M+ H]+ (protonated molecular ions). Searches were conducted using a mass accuracy of±100 ppm, and one missed cleavage site was allowed for each search.
RT-PCR
The concentration of RNA was accurately quantified by spectrophotometric measurement, and 1 µg total RNA was separated on 1% formaldehyde agarose gel to check their concentration and to monitor their integrity. Five hundred ng of total RNA was used in an RT-PCR system together with gene-specific primers. Control RT-PCR was performed with the same amount of total RNA using the primer pair specific to the tublin gene. Twenty microliter of each RT-PCR product was loaded on 0.8% (w/v) agarose gel to visualize the amplified cDNAs.
Results
Effects of different concentration of brassinolide on rice seedlings
Series concentrations of 0.1, 1, 10, 100, and 1000 nmol/L BL solution were separately added to the culture medium of the rice seedlings grown in a greenhouse for five days when their root length reached 1cm. It was found that the root growth was influenced by different concentrations of BL, which promoted or inhibited the rice root elongation, and the influence was concentration dependent. There was approximately 28% increase in root elongation at 0.1 nmol/L BL, and 65% root growth was inhibited at 1000 nmol/L BL as compared with the control, and these differences were significant (Table 1). The rice root was stained using PI and observed with confocal fluorescence microscope, which indicated that the cells of mature zone of the root had shorter length and wider width in 1000 nmol/L BL treatment than in the control. Fewer meristem layers in 1000 nmol/L BL treatment but more meristem layers in 0.1 nmol/L BL treatment were found as compared those in the control. A longer mature zone in that 0, 0.1 nmol/L BL treatment was observed as compared to that in the 100 nmol/L BL treatment. There were more cells outside the top of the root of BL treatments than that in the control (Fig. 1.).
2D-PAGE separation of the total proteins of rice root after BL treatments
The results showed that there was an opposite effect on the rice root growth between 0.1 nmol/L and 1000 nmol/L BL treatment. To find the mechanism of the regulation of BL on rice development, we analyzed the difference of the total proteins of the root treated with 0.1 and 1000 nmol/L BL using proteomic method.
The total protein was extracted from the root tissue and separated in the first dimension by IPG and in the second dimension by SDS-PAGE. Approximately, 800 protein spots were detected on Comas Blue-stained 2D gel. Analysis of those protein spots whose abundance was varied reproducibly greater than twofold was conducted by MALDI-TOF MS, which led to the identification of 33 different proteins (Table 2). Of the proteins identified, most had their known functions or sequences similar to those of the known proteins, whereas there are some novel peptides that have not been assigned by any functions. All proteins were categorized into classes based on their functions. The BR-regulated proteins interacted in signaling pathways, such as auxin, stress cell development, etc. (Table 2).
BR possibly affected the IAA release from the conjugate form in rice
Our proteomics results show that 1000 nmol/L BL treatment upregulated IAA amidohydrolase, but 0.1nmol/L BL inhibited IAA amidohydrolase (Fig. 2). The RT-PCR results of IAA amidohydrolase also showed that the RNA level of IAA amidohydrolase was decreased when treated with 0.1 nmol/L BL and increased when treated with 1000 nmol/L BL (Fig. 3).
To confirm the IAA content after BL treatments, we identified the free IAA level using HLPC (Table 3). The results showed that the free IAA level of 0.1 nmol/L BL-treated root was decreased by 45.7% lower than that of control, while the IAA level of 1000 nmol/L BL treatment was increased to 17.3% higher than that of control.
Discussion
Indole-3-acetic acid (IAA) is a signaling molecule that modulates division and elongation of plant cells (
Davies, 1995). Plants contain little free IAA, and most IAA is found conjugated to amino acids, peptides, sugars, or high molecular weight glycans. These conjugates have been implicated in such processes as storage, transport, and protection from oxidative degradation (
Cohen and Bandurski, 1982). Plants produce active IAA by de novo synthesis and by hydrolyzing IAA conjugates (
Bartel, 1997;
Normanly, 1997). IAA conjugation activity is widely distributed in the plant kingdom from mosses to angiosperms (
Sztein et al., 1995). IAA-conjugate hydrolases release free IAA from the conjugate form and thus are likely to play an important role in regulating free IAA levels. These hydrolases have been detected in bacteria and in a variety of plants (
Hall and Bandurski, 1986;
Kowalczyk and Bandurski, 1990;
Chou et al., 1996;
Bartel, 1997). Interestingly, the infection with
Plasmodiophora brassicae (which causes a clubroot disease) correlates with a dramatic increase in the rate of IAA–Asp hydrolysis (
Ludwig-Müller et al., 1996). This induction of one specific hydrolytic activity in response to a particular challenge suggests that various conjugate hydrolases might supply free IAA in response to a variety of needs. Several exogenous IAA conjugates mimic IAA (
Feung et al., 1977;
Hangarter et al., 1980;
Šoškić et al., 1995), suggesting that these conjugates are either auxins or hydrolyzed to release IAA. Bartel et al. cloned the
ILR1 gene and found that it was an amidohydrolase,
ILR1 catalyzed IAA-amino acid hydrolysis, thus released active indole-3-acetic acid from conjugates. The
ILR1 (IAA-Leu-resistant) mutant elongates roots at concentrations of IAA-Leu that inhibit wild-type root growth.
The activity of indole-3-acetamide (IAM) hydrolase found in various organs of rice plants is prominent in wild and cultivated rice. In order for the rice amidohydrolase to serve in auxin biosynthesis, high concentrations of substrate must be present endogenously. However, no endogenous IAM was detected from various organs, such as shoots, roots, calli, and young fruits of rice. Therefore, it is unlikely that the rice enzyme is involved in auxin biosynthesis via the IAM pathway. As the enzyme has dual functions as amidase and esterase, therefore, the rice amide hydrolase may serve to control IAA accumulation via hydrolysis of the endogenous esterified IAA.
Our results found that the IAA amidohydrolase decreased when treated with 0.01 nmol/L BL, but it increased when treated with 1000 nmol/L BL. The low concentration of IAA promoted the elongation of root; however, very high concentration of IAA increased the elongation of root. In the 1000 nmol/L BL treatment, the IAA amidohydrolase increased, and the pool IAA was almost activated. In addition, BL stimulated polar auxin transport and modified the distribution of endogenous (
Li, 2005), so the concentration of the activated IAA was very high with the phenotype of the root shortened and increased lateral roots when the rice seedling was treated with 1000 nmol/L BL. However, when the rice was treated with 0.01 nmol/L BL, the IAA amidohydrolase was low; therefore, there was a low level of activated IAA, and root elongation was promoted.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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