IC4R001-Proteomic-2003-12576669
Contents
Project Title
Characterization of Proteins Responsive to Gibberellin in the Leaf-Sheath of Rice (Oryza sativa L.) Seedling Using Proteome Analysis
The Background of This Project
- The phytohormone gibberellins (GAs) are essential endogenous regulators of plant growth and developmental processes including seed germination, stem elongation, leaf growth, cell division, flowering and fruit development. In recent years, significant progress has been made in under- standing the pathways involved in GA biosynthesis and on the mechanisms by which GA levels are regulated in plants. Despite this progress, the potential elements involved in GA perception and signal molecules that are transduced to control GA-regulated gene expression are still elusive.
In this study, the researchers report a systematic analysis aimed at establishing an effective approach to understand the molecular
basis for analyzing the GA controlled leaf-sheath elongation response in rice. For this, a proteome map of rice leaf-sheath was
constructed using 2-D PAGE, Edman sequencing and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry
(MALDI-TOF-MS)
Figure 1 (A) Dose dependent elongation of leaf sheath after treatment with GA 3 . Leaf sheaths excised from two-week-old rice seedlings grown in a 12 h light/12 h dark growth chamber at 25 °C were floated on Milli Q water containing 0.1, 1.0, 5.0, 10.0 or 50.0 m M GA 3 , and incubated under continuous light at 25 °C for 48 h.
Plant Culture & Treatment
- Oryza sativa L. cv. Nipponbare seedlings were grown under white fluorescent light (6000 lux, 12 h light period/d) at 25 °C. Three-cm-long leaf-sheath segments were cut from two-week-old rice seedlings, and floated on a 20 ml solution of GA 3 , with or without uniconazole p (Un-p), and ABA in sterile plastic petri dishes, and were incubated under a continuous light source in a growth chamber at 25 °C for various time periods as indicated.
Protein Extraction and 2-D PAGE
- A portion (500 mg) of leaf sheaths was homogenized in 1 ml of a homogenization buffer containing 20 m M Tris–HCl (pH 7.5), 250 m M sucrose, 10 m M EGTA, 1 m M phenylmethylsulfonyl fluoride, 1 m M dithiothreitol (DTT) and 1% (v/v) Triton X-100. The homogenates were centrifuged twice at 15000 rpm for 5 min and for 10 min in a TMA-4 rotor (Tomy, Tokyo, Japan). The supernatant was precipitated by 10% trichloroacetic acid on ice for 20 min, followed by centrifugation at 15000 rpm for 10 min.
- The pellet was suspended in 250 m l of a lysis buffer containing 8 M Urea, 2% Triton X-100, 2% ampholine (pI 3.5-10), 10% polyvinylpyrrolidone, and the supernatant (50 ( m l) was subjected to 2-D PAGE. The gel was stained with Coomassie brilliant blue (CBB). The isoelectric point and molecular mass of each protein were calibrated using 2-D PAGE standards (Bio-Rad, Richmond, CA, U.S.A.).
- The CBB stained gels were scanned using a flatbed scanner, and the data were analyzed using Image Master 2D Elite software (Amersham Pharmacia Biotech, Uppsala, Sweden).
Research Findings
- To assess the effective response for the promotion of rice leafsheath elongation by GA 3 , excised leaf-sheath segments of two-week-seedlings were treated with exogenous GA 3 for different time periods as described in Fig. 1. Exogenous application of GA 3 in amounts as low as 0.1 m M promoted second leaf-sheath elongation, and the effect was saturated at 5 mM after incubation for 48 h (Fig. 1A). To further examine the kinetics of GA 3 action on leaf-sheath elongation, a time-course experiment was conducted by treating leaf-sheath segments with 5 m M GA 3 for up to 72 h.
- The leaf sheath showed significant elongation within 6 h and peaked within 48 h of GA 3 treatment, indicating strongly the role of GAs in the elongation. This result showed that the elongation of the leaf-sheath is strongly stimulated by 5 m M GA 3 within 48h and this parameter was used in subsequent experiments.Though it has been well documented that GA promotes elongation growth by effecting cell division and expansion through activation of histone H1 kinase and cyclin genes, 21) physiological and genetic control of elongation has not yet been fully elucidated.
- To determine the proteins related to the GA 3 response in rice leaf-sheath, and to identify the various proteins contributing to the leaf-sheath elongation response, the proteins were compared by differential display analysis. They were extracted from leaf-sheaths treated in the presence or absence of GA 3. A large number of protein spots were found separated on 2-D PAGE gel stained with CBB. Most of the proteins from control and GA 3 -treated samples were separated with the same electrophoretic mobility on a 2-D PAGE, which was evident using internal protein markers, employed to calibrate pro- teins according to their molecular mass and isoelectric points.
- To precisely determine the function of calreticulin in rice tissues, the full-length cDNA for calreticulin 18) was introduced into rice in the sense and antisense orientation under the control of the CaMV 35S promoter in the pIG121-Hm vector Twenty independent lines of transgenic plants were regenerated and were confirmed by immunoblotting (Fig. 6D). The expression of calreticulin protein was more than double in calreticulin-sense transgenic rice leaf tissues as compared to control and antisense transgenic rice. Inter- estingly, the over-expression of calreticulin inhibited the callus regeneration and also the rate of seedling growth compared with the control and antisense rice.
Labs working on this Project
- Department of Molecular Biology, National Institute of Agrobiological Sciences; 2–1–2 Kannondai, Tsukuba, Ibaraki 305–8602, Japan.
