Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Proteomic Analysis of Peg-induced Drought Stress Responsive Protein in Terf1 Overexpressed Sugarcane (saccharum Officinarum) Leaves

M. Anisur Rahman
Bangladesh Sugarcane Research Institute, Ishurdi 6620, Pabna, Bangladesh

Lei Ren
Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Wei Wu
Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Yanchun Yan
Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract/ Executive Summary:

Drought is the major abiotic stress limiting sugarcane growth and productivity. ERF proteins regulate a variety of stress responses in plant. Overexpression of TERF1 can enhance the tolerance of transgenic sugarcane to drought stress. To improve the efficiency of sugarcane breeding, better understanding of the tolerance mechanism at molecular level is required. Two-dimensional gel electrophoresis (2-DE) coupled tandem mass spectrometry (MS/MS) analyses were conducted to compare the leaf proteome of the TERF1 OE and WT sugarcane plants to PEG stress. Using statistical program, 50 significantly differential protein spots were detected, of which 36 spots were identified by PMF and MS/MS fragmentation. Most of the identified proteins corresponded to metabolism, energy, protein synthesis, and disease/defense. Results implicated that the involvement of different metabolic pathways that may be activated in the TERF1 overexpressed transgenic sugarcane to cope with drought environment. Of the identified proteins, abundance of pentatricopeptide repeat (PPR) containing protein and peptidyl-prolyl cis-trans isomerase (PPIase) were decreased, but the abundance of vital proteins, such as metabolism protein (14-3-3 like protein), photosynthetic protein (RuBisCO large subunit, PEP carboxylase), ferredoxin, glyceraldehyde 3-phosphate dehydrogenase, elongation factor Tu, several small heat shock proteins, and peroxidases were increased. Analysis of protein properties showed that majority of the differentially abundant proteins associated with drought were stable, hydrophilic, and transmembrane proteins. Thus, the results of our study unravel the regulatory mechanism of TERF1 for drought stress tolerance of transgenic sugarcane and provide new insight into adaptation to osmotic stress through altering the expression of particular proteins.

Keywords: Proteomics, PEG stress, TERF1overexpression, Sugarcane, 2-DE, Mass spectrometry

Location: In Bangladesh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Sugarcane, Protein, Drought

Objectives:

The object of this study was to identify the proteins that altered due to overexpression of TERF1 gene to improve drought tolerance in transgenic sugarcane.

Methodology:

In our previous work, TERF1 overexpressed transgenic sugarcane plants were obtained through the Agrobacterium-mediated method. Full length of TERF1 complementary DNA (cDNA) was introduced into sugarcane cultivar Guitang 281 under the control of CaMV35S promoter, through Agrobacterium-mediated transformation. Wild-type (WT) plants were regenerated from non-transformed callus of the same cultivar under the same condition. Subsequently, plantlets were placed in the plastic pots with 25-cm width and 30-cm depth. Single plant of OE and WT lines were placed in each pot with three replications and kept in greenhouse. Drought-tolerant OE sugarcane lines were selected and the ectopic expression of TERF1 was checked by (RT)-PCR. For drought treatment, 7-month-old OE and WT sugarcane plants were exposed to polyethylene glycol (PEG 8000; 20 % w/v) by flushing 1-L PEG solutions to each pot every 5-day interval. Control pots were maintained under well-watered condition. After 21 days of treatment, the second leaves of OE and WT lines from drought stress conditions and normal conditions were harvested in three replicates, stored at −70 °C and used for protein extraction. Leaves for measuring chlorophyll and RWC were collected under drought stress conditions at the 7th, 14th, and 21st day from the second leaf of the same plant under drought stress conditions and normal conditions. Determination of Chlorophyll Content Leaf chlorophyll content was estimated following the method of Moran (1982). Briefly, second fully expanded leaves as described above were selected. Leaf disks with an area of 1 cm2 were cut by cork borer from the leaf blade. Three independent replicates of each treatment were selected and two disks of each leaf were made. The leaf disks were incubated in the dark at 4°C for 24 h in vials containing 5 mL N, N-dimethylformamide (DMF). Three-milliliter chlorophyll extraction was taken out for chlorophyll quantification. The chlorophyll concentration was quantified using a UV-Visible spectrophotometer (BioMate 3S, Thermo Scientific) at wavelengths of 647 and 664 nm (OD664 and OD647, respectively). Total chlorophyll content was calculated by the equation below and expressed in the unit microgram per square centimeter (μg/cm²). Chlt ¼ 7:04 OD₆₆₄ þ 20:27 OD₆₄₇. Basic Local Alignment Search Tools (BLAST) of NCBI (http://ncbi.nlm.nih.gov/blast/) was used to know the sequence of MS/MS-analyzed protein. Identified proteins were divided into functional class as described by Bevan et al. (1998), using Database for Annotation, Visualization and Integrated Discovery (http://david.abcc.ncifcrf.gov), ProtFun server (www.cbs.dtu.dk/services/ProtFun/), and UniProt database (www.uniprot.org) search. Physical and chemical properties, hydrophobicity, and transmembrane analysis of differentially-expressed proteins were done by Protparam, ProtScale, and TMpred tools of the Swiss Institute of Bioinformatics (www.expasy.ch/tools).

Source of Information: Plant Molecular Biology Reporter · June 2014

Article Link (Online Journal): DOI 10.1007/s11105-014-0784-3

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, this work provides new insight on transgene function underlying the defense mechanism of TERF1-overexpressed transgenic sugarcane in response to PEG-induced drought stress. However, further investigation for validating the proteins is needed to provide more information about the regulatory function of this transcription factor gene.

Knowledge Management: Journal